Nucleic acids for treatment of allergies

ABSTRACT

The present invention provides DNA vaccines for the treatment of allergies. The vaccines comprise the coding sequence for one or more allergenic epitopes, and preferably the full protein sequence, of the allergenic protein from which the epitope(s) is derived, fused inframe with the lumenal domain of the lysosomal associated membrane protein (LAMP) and the targeting sequence of LAMP. The vaccines allow for presentation of properly configured three dimensional epitopes for production of an immune response. The vaccines can be multivalent molecules, and/or can be provided as part of a multivalent vaccine containing two or more DNA constructs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation U.S. application Ser. No. 14/407,410,filed on May 6, 2015, which is a national stage of InternationalApplication No. PCT/US2012/042552, filed on Jun. 15, 2012.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the fields of molecular biology andmedicine. More specifically, the invention relates to nucleic acids foruse as DNA vaccines, and methods of using them to treat subjectssuffering from or susceptible to allergic reactions.

2. Discussion of Related Art

Allergy is a hypersensitivity disease characterized by the production ofIgE antibodies against an allergen, or allergy-causing molecule.Allergies affect more than 25% of the population. Allergens can enterthe body through many routes, including the respiratory tract, skincontact, ingestion, insect bite, or injection of a drug.

Allergy disease management comprises diagnosis and treatment. Allergistsdiagnose an allergy using a variety of techniques, such as a skin pricktest, radioallergosorbent-based techniques, ELISA, or provocation testto demonstrate allergen specific IgE and to identify the allergensource. Treatment of allergy most often falls into two categories:avoidance and dosing with anti-histamines. A third alternative, allergyimmunotherapy, requires that the patient receive weekly injectionsconsisting of small amounts of the offending allergens in order to helpthe immune system reeducate its response to the allergen.

The use and generation of allergen fusion proteins are well known in theart. For example, U.S. Pat. No. 7,566,456 teaches a fusion protein withIgE and IgG binding domains as well as encoding an allergen. Further, WO97/07218 teaches allergen-anti-CD32 fusion proteins for use in allergyimmunotherapy. Neither of these documents, however, teaches how theirrespective fusion protein interacts with T cells through antigenpresentation to induce or modify a Th1 response. Furthermore, there isno theoretical connection between directing the anti-CD32 containingvaccine to dendritic cells to effect a positive induction of Th1 cells.Both of these documents teach a composition that introduces an allergentherapeutically, such that the allergen can be found in the serum as anallergen-fusion protein.

It has been established by Toda et al., 2002 that a T cell epitope of anallergen, in this case a Cry J2 epitope located at amino acid 247-258,can be attached to a fusion protein and be used to conductallergy-specific immunotherapy. The specific composition described byToda et al., 2002 is the use of a DNA vaccine encoding the major CD4 Tcell epitope of Cry J2, located at amino acids 247-258, attached toclass II-associated invariant chain peptide (CLIP). CLIP contains alysosomal/endosomal trafficking sequence and contains a domain thatbinds to the peptide binding groove of MHC II. Toda et al., 2002 showsthat immunization with the Cry J2 peptide/CLIP DNA vaccine results inpriming a mouse to a predominantly Th1 response, characterized by higherIFN-gamma and IgG2a production. However, Toda et al. does not teach theintracellular targeting of the entire protein coding sequence of anallergen useful for conducting allergy-specific immunotherapy.

U.S. Pat. No. 6,982,326 and U.S. Pat. No. 6,090,386 describe nucleicacid sequences coding for the Cryptomeria japonica major pollenallergens Cry J1, Cry J2, Jun s I, and Jun v I, and fragments orpeptides thereof. The invention also provides purified Cry J1, Cry J2,Jun s I, and Jun v I, and at least one fragment thereof produced in ahost cell transformed with a nucleic acid sequence coding for Cry J1,Cry J2, Jun s I, and Jun v I, or at least one fragment thereof, andfragments of Cry J1, Cry J2, Jun s I, or Jun v I, or at least onefragment thereof, and fragments of Cry J1, Cry J2, Jun s I, or Jun v Iprepared synthetically. Cry J1, Cry J2, Jun s I, and Jun v I, andfragments thereof are disclosed as useful for diagnosing, treating, andpreventing Japanese cedar pollinosis. The invention also providesisolated peptides of Cry J1 and Cry J2. Peptides within the scope of theinvention comprise at least one T cell epitope, or preferably at leasttwo T cell epitopes of Cry J1 or Cry J2. The invention also pertains tomodified peptides having similar or enhanced therapeutic properties asthe corresponding naturally-occurring allergen or portion thereof buthaving reduced side effects. Methods of treatment or of diagnosis ofsensitivity to Japanese cedar pollens in an individual and therapeuticcompositions, and multi-peptide formulations comprising one or morepeptides of the invention are also provided. The invention does notteach how to combine the epitopes or allergens into a DNA vaccine withimmunostimulatory properties.

U.S. Pat. No. 7,547,440 and U.S. Pat. No. 7,112,329 identify the T-cellepitope site on a Japanese cypress (hinoki) pollen allergen molecule bystimulating a T-cell line established from a patient suffering fromJapanese cypress pollen allergy with an overlap peptide covering theprimary structure of the Japanese cypress pollen allergen. The peptideis useful in peptide-based immunotherapy for patients with spring treepollinosis including patients with Japanese cypress pollinosis havingcross reactivity with Japanese cypress pollen. The peptide is alsouseful for diagnosing spring tree pollinosis. The invention is limitedto diagnostics and polypeptide delivery of epitopes.

DNA vaccines have been developed as an alternative to traditional wholecell or whole virus vaccines. Generally speaking, DNA vaccines areengineered nucleic acids that include sequences encoding one or moreepitopes. The nucleic acids are delivered to cells, typically antigenpresenting cells (APCs), the nucleic acids are expressed, and theepitopes present on the expressed proteins are processed in theendosomal/lysosomal compartment, and ultimately presented on the surfaceof the cell. U.S. Pat. No. 5,633,234 to August et al. discloses andcharacterizes the endosomal/lysosomal targeting sequence of thelysosomal-associated membrane protein (LAMP). This patent identifiescritical residues in the C-terminal region of the protein, which arenecessary for targeting of the protein to the endosomal/lysosomalcompartment. The patent discloses that fusion of antigenic peptides tothe C-terminal LAMP targeting sequence can provide enhanced processingand presentation of epitopes for generation of an immune response.

In addition, U.S. patent application publication number 2004/0157307 toHarris et al. discloses the use of the LAMP lumenal domain as a“trafficking domain” to direct chimeric proteins expressed from DNAvaccines through one or more cellular compartments/organelles, such asthrough the lysosomal vesicular pathway. The chimeric proteins includethe lumenal domain of a LAMP polypeptide, an antigenic domain comprisinga peptide epitope sequence previously identified and selected from anantigen protein, a transmembrane domain, and an endosomal/lysosomaltargeting sequence.

DNA vaccines have been proposed as a treatment of allergic disease (Razet al., 1996; Hartl et al., 2004; Hsu et al., 1996; Crameri 2007; Weisset al., 2006). The underlying rationale is that allergen protein encodedby a DNA vaccine will preferentially activate the allergen-specific Th1cellular response with the production of interferons by APCs, naturalkiller (NK), and T cells, rather than the characteristic Th2-typeresponse, such as secretion of IL-4, IL-5, and IL-13, and the formationof IgE by B lymphocytes and the maturation and recruitment ofeosinophils in late-phase reactions. However, the mechanisms underlyingthe differential induction of the Th1 and Th2 T-cell phenotypes appearto involve a large number of factors, such as unique properties of thebacterial DNA of vaccine preparations, e.g., unmethylated and CpG DNAresidues, the cytokine milieu elicited by innate immunity, and thecellular trafficking properties of the allergens (Chen et al., 2001;Kaech et al., 2002). No invention or method has successfully addressedthe uncertainty of allergy treatment as conducted by delivery of nucleicacids encoding an allergen. Thus, to date such a method of allergytreatment has not been enabled. In addition, administration of DNAvaccines for the treatment of allergic disease has resulted in thesecretion of the allergen peptide into the extracellular environment,potentially leading to accidental induction of an allergic responsethrough activation of IgE.

SUMMARY OF THE INVENTION

The present invention provides nucleic acids (also referred to herein as“constructs”) that encode allergenic proteins, allergenic polypeptides,and allergenic peptides. The nucleic acids are designed for delivery toimmune cells and production of allergenic proteins, polypeptides, andpeptides within those cells. The encoded proteins, polypeptides, andpeptides have targeting sequences for targeting of the proteins to theMHC-II compartment for processing and display of one or more epitopes,resulting in an immune response to the epitope(s). In general, thenucleic acids comprise the following domains, which correlate to therespective domains of the encoded protein: a signal sequence domain; anintra-organelle stabilizing domain; an allergen domain; a transmembranedomain; and a cytoplasmic lysosome/endosome targeting domain.

Within the context of the encoded protein, the signal sequence isprovided to direct the encoded protein to the endoplasmic reticulum or alysosome. The intra-organelle stabilizing domain is a sequence that isdesigned to be proteolytically resistant and to protect the remainingportions of the protein, and in particular the allergen domain, fromdegradation prior to processing for epitope presentation by the cell. Inexemplary embodiments, the intra-organelle stabilizing domain is thelumenal domain of LAMP-1. The allergen domain comprises the sequence ofone or more allergenic epitopes that can serve to raise an immuneresponse in an animal in which the epitopes are presented. Typically,the allergen domain comprises one or more allergen proteins, although inembodiments, immunogenic polypeptide or peptide fragments of allergenicproteins can be used. In exemplary embodiments discussed below, theepitope is an epitope of a plant allergen. In the encoded proteins ofthe invention, the allergen domain does not include a signal peptide,such as the signal peptide(s) naturally occurring as part of theallergen protein(s). The allergen domain can comprise a singleallergenic protein, polypeptide, or peptide, or can comprise two or moreallergenic proteins, polypeptides, or peptides. Where two or moreallergens are present, each allergen can be from the same species/sourceor one or more can be from one or more different sources. Where two ormore allergens are present, they are coordinately expressed to providean equal number of copies of each coding region in the expressedprotein. The transmembrane domain can be any sequence that is suitablefor directing insertion and transfer of a protein through a membrane.Many such sequences are known in the art or can be easily designed. Thelysosome/endosome targeting domain can be any sequence that is capableof directing the peptide to a lysosome or endosome. Such sequences areknown in the art and are exemplified herein by the cytoplasmic tailsequence of LAMP-1.

As mentioned above, in preferred embodiments, the nucleic acids comprisean allergen domain that includes the entire allergenic coding sequencefor an allergenic protein, but lacks the coding sequence for theallergen's signal sequence. In some embodiments, the nucleic acids ofthe invention do not comprise the entire allergenic coding sequence, butinstead comprise only a sufficient amount of the coding sequence suchthat the encoded polypeptide, when expressed, is able to fold to achievethe natural three dimensional structure of at least one epitope presenton the polypeptide. As in constructs comprising an entire allergencoding sequence, where less than the entire coding sequence is present,the nucleic acids construct also lacks the coding sequence for anaturally-occurring signal peptide for the allergenic polypeptide orpeptide.

In preferred embodiments, the nucleic acid construct comprises thecoding sequences for multiple allergenic proteins, polypeptides, and/orpeptides in the allergen domain. Each allergen present can be from thesame source, each from a different source, or any combination thereof.

The nucleic acids, and thus the encoded proteins, polypeptides, andpeptides of the invention can be used in methods of treating subjects,and in particular animal subjects suffering from or potentiallydeveloping allergies. In general, a method of treating according to thepresent invention comprises administering a nucleic acid of theinvention to a subject in an amount sufficient to deliver the nucleicacid to one or more immune cells, and preferably to one or more antigenpresenting cells (APC) of the immune system. Once delivered, the nucleicacid is expressed, the encoded protein processed inside the cell, andthe epitope(s) displayed on the surface of the cell. The method oftreating can be considered a method of using the nucleic acids andproteins to provide a therapeutic or prophylactic immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a nucleic acid according to oneembodiment of the invention in which a single antigen comprising asingle epitope is provided in the allergen domain.

FIG. 2 shows a vector map of a nucleic acid according to the invention,in which the allergen domain comprises the CryJ2 allergen (an allergenfrom C. japonica), but without a signal sequence, inserted between humanLAMP N-terminal sequences (SS and ISOD) and human LAMP C-terminalsequences (TM and TG).

FIG. 3 is a schematic representation of a nucleic acid according to analternative embodiment of the invention, in which multiple epitopesequences of a single allergen are provided in the allergen domain.

FIG. 4 is a schematic representation of a nucleic acid according to analternative embodiment of the invention, in which multiple differentallergen sequences are provided in the allergen domain.

FIG. 5 shows a vector map of a nucleic acid according to the inventionin which the allergen domain comprises the allergen sequences (withoutsignal peptides) for the allergens CryJ1 (an allergen from C. japonica)and CryJ2 (an allergen from C. japonica).

FIG. 6A shows a vector map of a nucleic acid that includes three peanutallergens (AraH1, AraH2, and AraH3, all lacking signal sequences) in theallergen domain.

FIG. 6B shows a schematic of the protein encoded by the nucleic acid ofFIG. 6A.

FIG. 7 shows a vector map of a nucleic acid according to the presentinvention, depicting the absence of the naturally-occurring signalsequence for the CryJ1 allergen sequence. This particular construct isused in experiments detailed below to show the importance of removal ofthe natural signal sequence of allergen sequences.

FIG. 8 shows a vector map of a nucleic acid construct not encompassed bythe present invention, in which the CryJ2 allergen is encoded on aplasmid backbone, but in the absence of the SS, IOS, TM, and TG domains.This construct is used as a comparative control in experiments detailedbelow.

FIG. 9A and FIG. 9B show Western blots depicting expression ofconstructs according to the invention in 293 cells. FIG. 9A showsexpression of the CryJ1-CryJ2 combined allergens (see FIG. 5) and theCryJ2 allergen alone (see FIG. 2) in constructs according to theinvention, when assayed with anti-Cry J2 antibodies. FIG. 9B showsexpression of the CryJ1-CryJ2 combined allergens and the CryJ1 allergen(lacking its native signal sequence; see FIG. 7), when assayed withanti-CryJ1 antibodies. FIG. 9B further shows that expression of theCryJ1 allergen is not detectable in a construct in which the naturalsignal sequence for the CryJ1 allergen is not removed (vector map notshown).

FIG. 10A and FIG. 10B show line graphs depicting the effectiveness ofnucleic acid constructs according to the present invention as comparedto other constructs comprising allergen sequences. FIG. 10A shows that asignificant increase in IgG1 production and detection is seen as aresult of administration of the CryJ2-LAMP construct of the invention(see FIG. 2) as compared to a construct comprising a plasmid backbonefused to the CryJ2 coding sequence (see FIG. 8). FIG. 10B shows that asignificant increase in IgG2a production and detection is seen as aresult of administration of the CryJ2-LAMP construct of the invention(as per FIG. 10A) as compared to a construct comprising a plasmidbackbone fused to the CryJ2 coding sequence (as per FIG. 10A).

FIG. 11A and FIG. 11B depict bar graphs showing dosing effects of theCryJ2-LAMP construct in mice. FIG. 11A depicts IgG2a detection at 21days and 28 days post injection of the DNA vaccine at various amountsranging from 10 ug to 100 ug, as compared to injection of vector DNAalone. FIG. 11B depicts IgG1 detection at 21 days and 28 days postinjection of the DNA vaccine at various amounts ranging from 10 ug to100 ug, as compared to injection of vector DNA alone.

FIG. 12A and FIG. 12B depict bar graphs showing the effect on inductionof IL-4 and IFN-gamma in mouse spleen cultures treated with theCryJ2-LAMP construct of the invention as compared to vector alone. FIG.12A shows the effect of IL-4. FIG. 12B shows the effect of IFN-gamma.

FIG. 13A and FIG. 13B depict line graphs showing the effectiveness ofimmunization of previously sensitized mice with the CryJ2-LAMP DNAvaccine. FIG. 13A shows IgG1 titers over time. FIG. 13B shows IgG2atiters over time.

FIG. 14A and FIG. 14B depict bar graphs showing induction of IFN-g (FIG.14A) and IL-4 (FIG. 14B) in mouse spleen cell cultures.

FIG. 15 depicts a bar graph showing quantitation of circulating CryJ2protein in immunized mice.

FIG. 16A and FIG. 16B depict bar graphs of guinea pig data, showing IgG1detection (FIG. 16A) and IgG2 detection (FIG. 16B) for guinea pigsimmunized with the CryJ2-LAMP construct and challenged with recombinantCryJ2.

FIG. 17 depicts a bar graph showing the Anti-CryJ2 response in NewZealand white rabbits immunized with CryJ2-LAMP DNA vaccine during an 85day toxicology GLP safety study.

FIG. 18 depicts a Western blot showing co-expression of peanut allergensH1, H2, and H3 from a construct according to the present invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to various exemplary embodiments ofthe invention. It is to be understood that the following discussion ofexemplary embodiments is not intended as a limitation on the invention,as broadly disclosed herein. Rather, the following discussion isprovided to give the reader a more detailed understanding of certainaspects and features of the invention. The practice of the presentinvention employs, unless otherwise indicated, conventional molecularbiology, microbiology, and recombinant DNA techniques within the skillof those in the art. Such techniques are explained fully in theliterature known to the ordinary artisan in these fields, and thus neednot be detailed herein. Likewise, practice of the invention for medicaltreatment follows standard protocols known in the art, and thoseprotocols need not be detailed herein.

Before embodiments of the present invention are described in detail, itis to be understood that the terminology used herein is for the purposeof describing particular embodiments only, and is not intended to belimiting. Further, where a range of values is provided, it is understoodthat each intervening value, to the tenth of the unit of the lowerlimit, unless the context clearly dictates otherwise, between the upperand lower limits of that range is also specifically disclosed. Eachsmaller range between any stated value or intervening value in a statedrange and any other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included or excluded in the range,and each range where either, neither, or both limits are included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention. It is thus tobe understood that, where a range of values is presented, each valuewithin that range, and each range falling within that range, isinherently recited as well, and that the avoidance of a specificrecitation of each and every value and each and every possible range ofvalues is not an omission of those values and ranges, but instead is aconvenience for the reader and for brevity of this disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the term belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.The present disclosure is controlling to the extent it conflicts withany incorporated publication.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “an allergen” includes aplurality of such allergens and reference to “the sample” includesreference to one or more samples and equivalents thereof known to thoseskilled in the art, and so forth. Furthermore, the use of terms that canbe described using equivalent terms include the use of those equivalentterms. Thus, for example, the use of the term “subject” is to beunderstood to include the terms “animal”, “human”, and other terms usedin the art to indicate one who is subject to a medical treatment.

As used herein, the term “comprising” is intended to mean that theconstructs, compositions, and methods include the recited elementsand/or steps, but do not exclude other elements and/or steps.“Consisting essentially of, when used to define constructs,compositions, and methods, means excluding other elements and steps ofany essential significance to the recited constructs, compositions, andmethods. Thus, a composition consisting essentially of the elements asdefined herein would not exclude trace contaminants from the isolationand purification method and pharmaceutically acceptable carriers, suchas phosphate buffered saline, preservatives, and the like. “Consistingof means excluding more than trace elements of other ingredients andsubstantial method steps for administering the compositions of thisinvention. Embodiments defined by each of these transition terms arewithin the scope of this invention.

A “chimeric DNA” is an identifiable segment of DNA within a larger DNAmolecule that is not found in association with the larger molecule innature. Thus, when the chimeric DNA encodes a protein segment, thesegment coding sequence will be flanked by DNA that does not flank thecoding sequence in any naturally occurring genome. In the case where theflanking DNA encodes a polypeptide sequence, the encoded protein isreferred to as a “chimeric protein” (i.e., one having non-naturallyoccurring amino acid sequences fused together). Allelic variations ornaturally occurring mutational events do not give rise to a chimeric DNAor chimeric protein as defined herein.

As used herein, the terms “polynucleotide” and “nucleic acid molecule”are used interchangeably to refer to polymeric forms of nucleotides ofany length. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes, for example, single-,double-stranded and triple helical molecules, a gene or gene fragment,exons, introns, mRNA, tRNA, rRNA, ribozymes, antisense molecules, cDNA,recombinant polynucleotides, branched polynucleotides, aptamers,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A nucleic acid molecule mayalso comprise modified nucleic acid molecules (e.g., comprising modifiedbases, sugars, and/or internucleotide linkers).

As used herein, the term “peptide” refers to a compound of two or moresubunit amino acids, amino acid analogs, or peptidomimetics. Thesubunits may be linked by peptide bonds or by other bonds (e.g., asesters, ethers, and the like). The term “peptide” is used hereingenerically to refer to peptides (i.e., polyamino acids of from 2 toabout 20 residues), polypeptides (i.e., peptides of from about 20residues to about 100 residues), and proteins (i.e., peptides havingabout 100 or more residues).

As used herein, the term “amino acid” refers to either natural and/orunnatural or synthetic amino acids, including glycine and both D or Loptical isomers, and amino acid analogs and peptidomimetics. A peptideof three or more amino acids is commonly called an oligopeptide if thepeptide chain is short. While the term “protein” encompasses the term“polypeptide”, a “polypeptide” may be a less than a full-length protein.

The term “allergen” refers to any naturally occurring protein ormixtures of proteins that have been reported to induce allergic, i.e.,IgE mediated reactions upon their repeated exposure to an individual. Anallergen is any compound, substance, or material that is capable ofevoking an allergic reaction. Allergens are usually understood as asubcategory of antigens, which are compounds, substances, or materialscapable of evoking an immune response. For carrying out the invention,the allergen may be selected, among other things, from natural or nativeallergens, modified natural allergens, synthetic allergens, recombinantallergens, allergoids, and mixtures or combinations thereof. Ofparticular interest are allergens that are capable of causing anIgE-mediated immediate type hypersensitivity.

Examples of naturally occurring allergens include pollen allergens(e.g., tree, weed, herb and grass pollen allergens), mite allergens(from e.g. house dust mites and storage mites), insect allergens (e.g.,inhalant, saliva- and venom origin allergens), animal allergens frome.g. saliva, hair and dander from animals (e.g. dog, cat, horse, rat,mouse, etc.), fungi allergens and food allergens. The allergen may be inthe form of an allergen extract, a purified allergen, a modifiedallergen or a recombinant allergen or a recombinant mutant allergen, anallergen fragment above 30 amino acids or any combination thereof.

In terms of their chemical or biochemical nature, allergens canrepresent native or recombinant proteins or peptides, fragments ortruncated versions of native or recombinant proteins or peptides, fusionproteins, synthetic compounds (chemical allergens), synthetic compoundsthat mimic an allergen, or chemically or physically altered allergens,such as allergens modified by heat denaturation.

The classification of an allergen as a major allergen can be subject toseveral tests. An allergen is commonly classified as a major allergen ifat least 25% of patients show strong IgE binding (score 3) and at leastmoderate binding (score 2) from 50% of the patients, the binding beingdetermined by an CRIE (Crossed Radio Immune Electrophoresis) (CRIEStrong binding, i.e., visible IgE-binding on an X-ray film after oneday; CRIE Moderate binding, i.e., binding after 3 days; CRIE Weakbinding, i.e., binding after 10 days). Strong IgE binding from at least10% of the patients classifies the allergen as an Intermediate allergenand clearly specific binding from less than 10% of the patientsclassifies it as a Minor allergen. Other methods may also be used indetermining the IgE binding of for instance IgE-blots.

An “epitope” is a structure, usually made up of a short peptide sequenceor oligosaccharide, that is specifically recognized or specificallybound by a component of the immune system. T-cell epitopes havegenerally been shown to be linear oligopeptides. Two epitopes correspondto each other if they can be specifically bound by the same antibody.Two epitopes correspond to each other if both are capable of binding tothe same B cell receptor or to the same T cell receptor, and binding ofone antibody to its epitope substantially prevents binding by the otherepitope (e.g., less than about 30%, preferably, less than about 20%, andmore preferably, less than about 10%, 5%, 1%, or about 0.1% of the otherepitope binds).

As used herein, two nucleic acid coding sequences “correspond” to eachother if the sequences or their complementary sequences encode the sameamino acid sequences.

As used herein, a polynucleotide or polynucleotide region (or apolypeptide or polypeptide region) which has a certain percentage (forexample, at least about 50%, at least about 60%), at least about 70%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 99%) of “sequence identity” to another sequencemeans that, when maximally aligned, manually or using software programsroutine in the art, that percentage of bases (or amino acids) are thesame in comparing the two sequences.

Two nucleotide sequences are “substantially homologous” or“substantially similar” when at least about 50%, at least about 60%, atleast about 70%, at least about 75%, and preferably at least about 80%,and most preferably at least about 90 or 95% of the nucleotides matchover the defined length of the DNA sequences. Similarly, two polypeptidesequences are “substantially homologous” or “substantially similar” whenat least about 40%, at least about 50%), at least about 60%, at leastabout 66%, at least about 70%, at least about 75%, and preferably atleast about 80%, and most preferably at least about 90 or 95% or 98% ofthe amino acid residues of the polypeptide match over a defined lengthof the polypeptide sequence. Sequences that are substantially homologouscan be identified by comparing the sequences using standard softwareavailable in sequence data banks. Substantially homologous nucleic acidsequences also can be identified in a Southern hybridization experimentunder, for example, stringent conditions as defined for that particularsystem. Defining appropriate hybridization conditions is within theskill of the art. For example, stringent conditions can be:hybridization at 5×SSC and 50% formamide at 42° C., and washing at0.1×SSC and 0.1% sodium dodecyl sulfate at 60° C.

“Conservatively modified variants” of domain sequences also can beprovided. With respect to particular nucleic acid sequences, the termconservatively modified variants refers to those nucleic acids thatencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical sequences. Specifically, degenerate codon substitutions can beachieved by generating sequences in which the third position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer, et al., 1991, Nucleic Acid Res. 19: 5081;Ohtsuka, et al., 1985, J. Biol. Chem. 260: 2605-2608; Rossolini et al.,1994, Mol. Cell. Probes 8: 91-98).

The term “biologically active fragment”, “biologically active form”,“biologically active equivalent”, and “functional derivative” of awild-type protein, means a substance that possesses a biologicalactivity that is at least substantially equal (e.g., not significantlydifferent from) the biological activity of the wild type protein asmeasured using an assay suitable for detecting the activity. Forexample, a biologically active fragment comprising a trafficking domainis one which can co-localize to the same compartment as a full lengthpolypeptide comprising the trafficking domain.

A cell has been “transformed”, “transduced”, or “transfected” byexogenous or heterologous nucleic acids when such nucleic acids havebeen introduced inside the cell. Transforming DNA may or may not beintegrated (covalently linked) with chromosomal DNA making up the genomeof the cell. In prokaryotes, yeast, and mammalian cells for example, thetransforming DNA may be maintained on an episomal element, such as aplasmid. In a eukaryotic cell, a stably transformed cell is one in whichthe transforming DNA has become integrated into a chromosome so that itis inherited by daughter cells through chromosome replication. Thisstability is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the transforming DNA. A “clone” is a population ofcells derived from a single cell or common ancestor by mitosis. A “cellline” is a clone of a primary cell that is capable of stable growth invitro for many generations (e.g., at least about 10).

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo.

As used herein, a “viral vector” refers to a virus or viral particlethat comprises a polynucleotide to be delivered into a host cell, eitherin vivo, ex vivo, or in vitro. Examples of viral vectors include, butare not limited to, adenovirus vectors, adeno-associated virus vectors,retroviral vectors, and the like. In aspects where gene transfer ismediated by an adenoviral vector, a vector construct refers to thepolynucleotide comprising the adenovirus genome or part thereof, and aselected, non-adeno viral gene, in association with adenoviral capsidproteins.

As used herein, a “nucleic acid delivery vector” is a nucleic acidmolecule that can transport a polynucleotide of interest into a cell.Preferably, such a vector comprises a coding sequence operably linked toan expression control sequence. However, a polynucleotide sequence ofinterest does not necessarily comprise a coding sequence. For example,in one aspect, a polynucleotide sequence of interest is an aptamer whichbinds to a target molecule. In another aspect, the sequence of interestis a complementary sequence of a regulatory sequence that binds to aregulatory sequence to inhibit regulation of the regulatory sequence. Instill another aspect, the sequence of interest is itself a regulatorysequence (e.g., for titrating out regulatory factors in a cell).

As used herein, a “nucleic acid delivery vehicle” is defined as anymolecule or group of molecules or macromolecules that can carry insertedpolynucleotides into a host cell (e.g., such as genes or gene fragments,antisense molecules, ribozymes, aptamers, and the like) and that occursin association with a nucleic acid delivery vector as described above.

As used herein, “nucleic acid delivery” or “nucleic acid transfer”refers to the introduction of an exogenous polynucleotide (e.g., such asa transgene) into a host cell, irrespective of the method used for theintroduction. The introduced polynucleotide may be stably or transientlymaintained in the host cell. Stable maintenance typically requires thatthe introduced polynucleotide either contains an origin of replicationcompatible with the host cell or integrates into a replicon of the hostcell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclearor mitochondrial chromosome.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or translated intopeptides, polypeptides, or proteins. If the polynucleotide is derivedfrom genomic DNA, expression may include splicing of the mRNAtranscribed from the genomic DNA.

As used herein, “under transcriptional control” or “operably linked”refers to expression (e.g., transcription or translation) of apolynucleotide sequence which is controlled by an appropriatejuxtaposition of an expression control element and a coding sequence. Inone aspect, a DNA sequence is “operatively linked” to an expressioncontrol sequence when the expression control sequence controls andregulates the transcription of that DNA sequence.

As used herein, “coding sequence” is a sequence which is transcribed andtranslated into a polypeptide when placed under the control ofappropriate expression control sequences. The boundaries of a codingsequence are determined by a start codon at the 5′ (amino) terminus anda translation stop codon at the 3′ (carboxyl) terminus. A codingsequence can include, but is not limited to, a prokaryotic sequence,cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g.,mammalian) DNA, and even synthetic DNA sequences. A polyadenylationsignal and transcription termination sequence will usually be located 3′to the coding sequence.

As used herein, a “genetic modification” refers to any addition to ordeletion or disruption of a cell's normal nucleotide sequence. Anymethod that can achieve the genetic modification of APCs are within thespirit and scope of this invention. Art recognized methods include viralmediated gene transfer, liposome mediated transfer, transformation,transfection and transduction, e.g., viral-mediated gene transfer suchas the use of vectors based on DNA viruses such as adenovirus,adeno-associated virus and herpes virus, as well as retroviral basedvectors.

As used herein, “the lysosomal/endosomal compartment” refers tomembrane-bound acidic vacuoles containing LAMP molecules in themembrane, hydrolytic enzymes that function in antigen processing, andWIC class II molecules for antigen recognition and presentation. Thiscompartment functions as a site for degradation of foreign materialsinternalized from the cell surface by any of a variety of mechanismsincluding endocytosis, phagocytosis, and pinocytosis, and ofintracellular material delivered to this compartment by specializedautolytic phenomena (see, for example, de Duve, Eur. J. Biochem. 137:391, 1983). The term “endosome” as used herein encompasses a lysosome.

As used herein, a “lysosome-related organelle” refers to any organellethat comprises lysozymes and includes, but is not limited to, MIIC, CUV,melanosomes, secretory granules, lytic granules, platelet-densegranules, basophil granules, Birbeck granules, phagolysosomes, secretorylysosomes, and the like. Preferably, such an organelle lacks mannose6-phosphate receptors and comprises LAMP, but might or might notcomprise an MHC class II molecule. For reviews, see, e.g., Blott andGriffiths, Nature Reviews, Molecular Cell Biology, 2002; DellAngelica,et al., The FASEB Journal 14: 1265-1278, 2000.

As used herein a “LAMP polypeptide” refers to LAMP-1, LAMP-2,CD63/LAMP-3, DC-LAMP, or any lysosomal associated membrane protein, orhomologs, orthologs, variants (e.g., allelic variants) and modifiedforms (e.g., comprising one or more mutations, either naturallyoccurring or engineered). In one aspect, a LAMP polypeptide is amammalian lysosomal associated membrane protein, e.g., such as a humanor mouse lysosomal associated membrane protein. More generally, a“lysosomal membrane protein” refers to any protein comprising a domainfound in the membrane of an endosomal/lysosomal compartment orlysosome-related organelle and which further comprises a lumenal domain.

As used herein, “targeting” denotes the polypeptide sequence thatdirects a chimeric protein of the invention to a preferred site, such asa cellular organelle or compartment where antigen processing and bindingto MHC II occurs. As such, a “targeting domain” refers to a series ofamino acids that are required for delivery to a cellularcompartment/organelle. Preferably, a targeting domain is a sequence thatbinds to an adaptor or AP protein (e.g., such as an AP1, AP2, or AP3protein). Exemplary targeting domain sequences are described inDellAngelica, 2000, for example.

As used herein, in vivo nucleic acid delivery, nucleic acid transfer,nucleic acid therapy, and the like, refer to the introduction of avector comprising an exogenous polynucleotide directly into the body ofan organism, such as a human or non-human mammal, whereby the exogenouspolynucleotide is introduced into a cell of such organism in vivo.

As used herein, the term in situ refers to a type of in vivo nucleicacid delivery in which the nucleic acid is brought into proximity with atarget cell (e.g., the nucleic acid is not administered systemically).For example, in situ delivery methods include, but are not limited to,injecting a nucleic acid directly at a site (e.g., into a tissue, suchas a tumor or heart muscle), contacting the nucleic acid with cell(s) ortissue through an open surgical field, or delivering the nucleic acid toa site using a medical access device such as a catheter.

As used herein, the terms “isolated” and “purified” are used at timesinterchangeably to mean separated from constituents, cellular andotherwise, in which the polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, are normally associated with in nature.For example, with respect to a polynucleotide, an isolatedpolynucleotide is one that is separated from the 5′ and 3′ sequenceswith which it is normally associated in the chromosome. As is apparentto those of skill in the art, a non-naturally occurring polynucleotide,peptide, polypeptide, protein, antibody, or fragments thereof, does notrequire “isolation” to distinguish it from its naturally occurringcounterpart. Furthermore, the terms “isolated” and “purified” do notimply total isolation and total purity. These terms are used to denoteboth partial and total purity from some or all other substancesnaturally found in association with the polynucleotide, etc. Thus, theseterms can mean isolation or purification from one naturally associatedsubstance (e.g., isolation or purification of DNA from RNA), isolationor purification from other substances of the same general class ofmolecule (e.g., a particular protein showing 20% purity as compared toall proteins in a sample), or any combination. Isolation andpurification can mean any level from about 1% to about 100%, including100%. As such, an “isolated” or “purified” population of cells issubstantially free of cells and materials with which it is associated innature. By substantially free or substantially purified APCs is meant atleast 50% of the population of cells are APCs, preferably at least 70%,more preferably at least 80%, and even more preferably at least 90% freeof non-APCs cells with which they are associated in nature. Of course,those of skill in the art will recognize that all specific values,including fractions of values, are encompassed within these rangeswithout the need for each particular value to be listed herein. Eachvalue is not specifically disclosed for the sake of brevity; however,the reader is to understand that each and every specific value isinherently disclosed and encompassed by the invention.

As used herein, a “target cell” or “recipient cell” refers to anindividual cell or cell which is desired to be, or has been, a recipientof exogenous nucleic acid molecules, polynucleotides, and/or proteins.The term is also intended to include progeny of a single cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic or total DNA complement) to the original parent cell due tonatural, accidental, or deliberate mutation. A target cell may be incontact with other cells (e.g., as in a tissue) or may be foundcirculating within the body of an organism.

The term “antigen presenting cell” or “APC” as used herein intends anycell that presents on its surface an antigen in association with a majorhistocompatibility complex molecule, or portion thereof, or,alternatively, one or more non-classical MEW molecules, or a portionthereof. Examples of suitable APCs are discussed in detail below andinclude, but are not limited to, whole cells such as macrophages,dendritic cells, B cells, hybrid APCs, and foster antigen presentingcells.

As used herein an “engineered antigen-presenting cell” refers to anantigen-presenting cell that has a non-natural molecular moiety on itssurface. For example, such a cell may not naturally have a co-stimulatoron its surface or may have additional artificial co-stimulator inaddition to natural co-stimulator on its surface, or may express anon-natural class II molecule on its surface.

As used herein, the term “immune effector cells” refers to cells thatare capable of binding an antigen and that mediate an immune response.These cells include, but are not limited to, T cells, B cells,monocytes, macrophages, NK cells, and cytotoxic T lymphocytes (CTLs),for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, orother infiltrates.

As used herein, the terms “subject” and “patient” are usedinterchangeably to indicate an animal for which the present invention isdirected. The term animal is to be understood to include humans andnon-human animals; where a distinction between the two is desired, theterms human and/or non-human animal are used. In embodiments, thesubject or patient is a vertebrate, preferably a mammal, more preferablya human. Mammals include, but are not limited to, murines, simians,humans, farm animals (e.g., bovines, ovines, porcines), sport animals(e.g. equines), and pets (e.g., canines and felines).

Clinical allergy symptoms are known to those of skill in the art, and anexhaustive listing herein is not required. Non-limiting examples includerhinitis, conjunctivitis, asthma, urticaria, eczema, which includesreactions in the skin, eyes, nose, upper and lower airways with commonsymptoms such as redness and itching of eyes and nose, itching and runnynose, coaching, wheezing, shortness of breath, itching, and swelling oftissue.

Examples of “immunological in vivo tests” are Skin Prick Test (SPT),Conjunctival Provocation Test (CPT), Bronchial Challenge with Allergen(BCA), and various clinical tests in which one or more allergy symptomsis monitored. See, for example, Haugaard et al., J Allergy Clin Immunol,Vol. 91, No. 3, pp 709-722, March 1993.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers known in theart, such as a phosphate buffered saline solution, water, and emulsions,such as an oil/water or water/oil emulsion, and various types of wettingagents. The compositions also can include stabilizers and preservatives.For examples of carriers, stabilizers and adjuvants, see MartinRemington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton (1975)).

As used herein, a “therapeutically effective amount” is used herein tomean an amount sufficient to prevent, correct, and/or normalize anabnormal physiological response. In one aspect, a “therapeuticallyeffective amount” is an amount sufficient to reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant feature of pathology, such asfor example, size of a tumor mass, antibody production, cytokineproduction, fever or white cell count, or level of histamine.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies (e.g., bispecific antibodies). An“antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen. Exemplary antibody moleculesare intact immunoglobulin molecules, substantially intact immunoglobulinmolecules, and those portions of an immunoglobulin molecule thatcontains the paratope, including Fab, Fab′, F(ab′)₂, and F(v) portions,which portions are preferred for use in the therapeutic methodsdescribed herein.

The term “oromucosal administration” refers to a route of administrationwhere the dosage form is placed under the tongue or anywhere else in theoral cavity to allow the active ingredient to come in contact with themucosa of the oral cavity or the pharynx of the patient in order toobtain a local or systemic effect of the active ingredient. An exampleof an oromucosal administration route is sublingual administration. Theterm “sublingual administration” refers to a route of administrationwhere a dosage form is placed underneath the tongue in order to obtain alocal or systemic effect of the active ingredient. As used herein, theterm “intradermal delivery” means delivery of the vaccine to the dermisin the skin. However, the vaccine will not necessarily be locatedexclusively in the dermis. The dermis is the layer in the skin locatedbetween about 1.0 and about 2.0 mm from the surface in human skin, butthere is a certain amount of variation between individuals and indifferent parts of the body. In general, it can be expected to reach thedermis by going 1.5 mm below the surface of the skin. The dermis islocated between the stratum corneum and the epidermis at the surface andthe subcutaneous layer below. Depending on the mode of delivery, thevaccine may ultimately be located solely or primarily within the dermis,or it may ultimately be distributed within the epidermis and the dermis.

As used herein, the term “prevent” in the context of allergyimmunotherapy, allergy treatment, or other terms that describe anintervention designed for an allergy patient, means the prevention of anIgE response in at least 20% of all patients. The term “prevent” doesnot mean total prevention from developing an IgE mediated disease in allpatients, and such a definition is outside the scope of the presentinvention for treating allergy through a mechanism that reduces allergysymptoms, and is inconsistent with the use of the term in the art. It iswell known to those skilled in the art of allergy immunotherapy thatallergy treatments are not 100% effective in 100% of patients, and assuch an absolute definition of “prevent” does not apply within thecontext of the present invention. The art-recognized concept ofprevention is contemplated by the present invention.

The present invention provides polynucleic acids, polyaminoacids, andmethods of treating subjects in need of the polynucleic acids andpolyaminoacids. Broadly speaking, the polynucleic acids can be thoughtof as nucleic acid (e.g., DNA, RNA) vaccines for the intracellularproduction of allergenic sequences (polyaminoacids) that elicit aprotective immune response within the body of the subject to whom thepolynucleic acid is administered. The polynucleic acids, whenadministered, preferentially evoke a cell-mediated immune response viathe MHC-II pathway and production of IgG antibodies by activating anallergen-specific T-helper type 1 (Th1) cellular response with theproduction of interferons by APCs, NK cells, and T cells rather than aTh2-type response, which involves production of IgE antibodies,granulocytes (e.g., eosinophils), and other substances. To an extent,both an MHC-II and an MHC-I response can be generated; however, theinvention provides a response that is primarily or substantially anMHC-II response. Preferably, the nucleic acids do not encode anantibiotic resistance gene.

The invention is based, at least in part, on the recognition that acombination of certain structural, and thus functional, elementsprovides advantageous properties to the nucleic acid vaccines and theencoded allergens, and allows for allergy treatment methods that satisfyunmet needs in the art. In the various embodiments of the invention,which are intended to be understood as standing alone as independentembodiments and as embodiments that combine two or more features of theindependent embodiments, the combinations include the use of a lysosomaltrafficking domain to direct allergen amino acid sequences to lysosomeswith WIC II proteins. Doing so allows for predominantly an IgG responseas opposed to an IgE response to the allergen sequences. Yet further,independent embodiments or combinations of embodiments provideconstructs containing a sufficient length of a nucleic acid sequence toencode an amino acid sequence that provides a naturally-occurringthree-dimensional structure of an epitope. In preferred embodiments, thenucleic acid sequence provides/encodes the full-length allergen codingsequence, but which lacks any naturally-occurring signal peptidesequence associated with the allergen sequence. In other embodiments,the nucleic acid sequence encodes at least one allergenic region of anallergen, but not the full-length allergen protein (and also lacking thesignal sequence, if one was naturally present). Although it isrecognized in the art that an immune response can be generated againstthe primary sequence of an epitope, the present invention recognizesthat nucleic acid vaccines for the production of an MHC-II immuneresponse to encoded epitopes preferably uses nucleic acid constructsthat encode enough sequence data to produce a correct three-dimensionalpeptide structure in the region comprising an allergenic epitope, atleast at the time when the allergenic sequence is delivered to alysosome for processing. While not being limited to any particularmolecular theory, it is believed that delivery of a properlythree-dimensionally folded protein, polypeptide, or peptide to anendosome improves processing and presentation of allergenic epitopes foran immune response.

As yet another example of an embodiment that can be implemented, aloneor as part of a combination of embodiments, the expression of multipleallergens from a single construct is provided. To date, it has not beenshown that a nucleic acid vaccine that is protective against an allergencan be effectively produced and used. The present invention not onlyprovides an effective nucleic acid vaccine against an allergen, butfurther provides an effective nucleic acid vaccine against multipleallergens at the same time. The allergens can be allergens from the samesource (e.g., a single plant), or can be allergens from two or moresources (e.g., a tree, a flower, a food, etc.). As above, thefull-length allergen sequences can be used (lacking anynaturally-associated signal sequence for the allergen), or allergenicportions can be used. In constructs comprising multiple allergensequences, any mixture of full-length or truncated allergen sequencescan be used. Further, as with other embodiments, it is preferred thatnaturally-occurring signal sequence for each allergen sequence beremoved (i.e., the naturally-occurring signal sequences for eachallergen sequence are not present in the constructs).

Although the use of signal sequences for the independent allergenicsequences within the allergen domain has been found to be detrimental tothe function of the nucleic acid construct, it has been found that theuse of signal sequence region or domain within the nucleic acid vaccineconstructs is an important feature. As such, in embodiments, the nucleicacid vaccine includes at least one signal sequence within the signalsequence domain to direct the encoded peptide to and through a membrane.Although the amino acid sequence of the signal sequence may vary fromconstruct to construct, and any known signal sequence can be selected,it has been found that in preferred embodiments, the signal sequence ispresent and provided in-frame with the coding sequence of the allergensequence(s). The use of a single signal sequence is adequate to directthe entire encoded chimeric protein to and through a membrane. As such,signal sequences for each allergen sequence are not necessary and, infact, have been found to be detrimental to proper localization,processing, and expression of allergen epitopes on immune cell surfaces.

And further, in specific embodiments and in combinations of embodiments,it has been found that sequestration, or physical protection, ofallergen sequences during the transfer of the polypeptide from thecytoplasm to the endosome, including the time in the endosome prior tocleavage of the polypeptide into units for presentation at the cellsurface, can be an important factor in providing a useful nucleic acidvaccine according to the invention. As such, in general, the inventionincludes a construct that comprises an intra-organelle stabilizingdomain (IOSD) to protect allergen sequences.

The nucleic acid of the invention comprises at least the followingdomains: a signal sequence domain; an intra-organelle stabilizingdomain; an allergen domain (which can comprise a single allergen or twoor more allergens, each comprising one or more allergenic epitopes); atransmembrane domain; and a cytoplasmic lysosome/endosome targetingdomain. The various domains are present on a single chimeric orengineered nucleic acid. The various domains can be combined in anylinear order using techniques known and widely practiced in the art. Inpreferred embodiments, the domains are combined and arranged such thatthey comprise a single open reading frame encoding a chimeric protein,the open reading frame being operably linked to transcriptional elementssufficient for expression of the chimeric protein. The nucleic acid thuscan be an expression vector, such as a plasmid, phagemid, viral vector,or the like. Preferably, the nucleic acid comprises transcriptionalelements suitable for expression in mammalian cells, such as humancells. Such expression vector elements and expression vectors are knownand widely used in the art, as exemplified by U.S. patent applicationpublication number 2004/0157307, which is incorporated herein byreference. A non-limiting example of a plasmid backbone for use increating nucleic acid constructs according to the invention is referredto at times herein as a “pITI” plasmid, the sequence of which isprovided as SEQ ID NO: 1.

Three exemplary configurations of the nucleic acid of the invention aredepicted schematically in FIGS. 1, 3, and 4, respectively. FIG. 1 showsa sequential arrangement of domains in which a single allergencomprising a single epitope is included in the encoded chimeric protein.FIG. 3 shows a sequential arrangement of domains in which multipledifferent epitopes of a single allergen are included in the encodedchimeric protein within the allergen domain. The two epitopes arearranged such that they are in the same reading frame and are thus bothproduced as part of the chimeric protein. Those of skill in the art willimmediately recognize that three or more epitopes can be provided in thesame reading frame within the epitope domain using standard molecularbiology techniques. FIG. 4 shows a sequential arrangement of domains inwhich two different allergens are present in the allergen domain. Ofcourse, the skilled artisan will recognize that each allergen sequencecan contain one or multiple allergenic epitopes. Based on these threeschematic representations of embodiments of the nucleic acids of theinvention, the reader will immediately recognize that any number ofallergens, from any number of sources, and containing any number ofepitopes, can be included within the allergen domain, and can be linkedin-frame using standard molecular biology techniques.

FIG. 2 depicts a vector map of a nucleic acid according to oneembodiment of the invention (“pITI-CRY J2-LAMP”; also referred to hereinat times as “CRYJ2-LAMP”), which generally relates to the embodiment ofthe invention depicted schematically in FIG. 1. The vector or deliveryvehicle includes a plasmid backbone with a pUC origin of replication andvarious transcription and expression elements for production of theencoded protein. More specifically, it includes the sequence of the pITIbackbone (SEQ ID NO: 1). It is to be noted that the nucleic acidconstruct does not include an antibiotic resistance gene, in accordancewith preferred embodiments of the invention. The nucleic acid furthercomprises sequences for the encoded protein, which comprises anN-terminal region of the human LAMP protein, which includes a signalsequence and an intra-organelle stabilizing domain. The nucleic acidfurther provides sequences for the encoded protein that comprises theCryJ2 allergen sequence (lacking its signal sequence) fused in-frame tothe N-terminal region of the LAMP protein. The nucleic acid furtherincludes sequences encoding a portion of the C-terminal region of thehuman LAMP protein, which includes a transmembrane region and atargeting region. The coding region for the CRY J2-LAMP chimeric proteinsequence is provided as SEQ ID NO:2. The amino acid sequence for the CRYJ2-LAMP chimeric protein is provided as SEQ ID NO:3.

In exemplary embodiments, the invention also relates to nucleic acidconstructs for the delivery and expression of other allergens of C.japonica, including the CryJ1 allergen. Using the same plasmid backbone,a pITI-CRYJ1-LAMP construct has been created. The chimeric protein canelicit an WIC II type immune response. The coding region for thepITI-CRYJ1-LAMP construct is presented as SEQ ID NO:4. The amino acidsequence for the CRY J1-LAMP chimeric protein is provided as SEQ IDNO:5.

As shown in FIGS. 3 and 4, the allergen domain can include an allergenhaving multiple allergenic epitopes, or can include multiple allergens(each having one or more allergenic epitopes). FIG. 5 depicts a vectormap of a particular exemplary embodiment of a nucleic acid construct inwhich the allergen domain includes two allergenic sequences. In thisexemplary embodiment, the allergen domain contains the CryJ1 and CryJ2allergens (each lacking its natural signal sequence) of C. japonicafused in-frame and fused at the N-terminal end with a LAMP signalsequence domain and intra-organelle stabilizing domain. The CryJ1-CryJ2sequences are also fused at the C-terminal end with a LAMP transmembranedomain and targeting domain. The full nucleotide sequence of the codingregion for the chimeric protein is presented as SEQ ID NO:6. The fullamino acid sequence of the encoded chimeric protein is presented as SEQID NO:7, in which: residues 1-27 represent the signal sequence for thechimeric protein; residues 28-380 represent the intra-organellestabilizing domain (sequence taken from human LAMP); residues 381 and382 represent a linker; residues 383-735 represent the coding region ofthe CryJ1 (without its signal sequence); residues 736-741 represent alinker region; residues 742-1232 represent the coding region for theCryJ2 allergen; residues 1233-1234 represent a linker region; residues1235-1258 represent the transmembrane and targeting domain; and residues1259-1270 represent additional C-terminal residues.

The nucleic acid constructs of the invention are essentially limitlessin the number of allergens that can be coordinately produced. As such,two, three, four, five, six, ten, twenty, or more different allergens(from the same or a mixture of different sources) can be included in thenucleic acid constructs of the invention. FIG. 6A presents a vector mapof another exemplary nucleic acid according to an embodiment of theinvention. The vector or delivery vehicle includes a plasmid backbonewith a pUC origin of replication and various transcription andexpression elements for production of the encoded protein. The backbonecan be, but is not necessarily, the pITI backbone of SEQ ID NO: 1. Thenucleic acid further comprises sequences for the encoded protein, whichcomprises an N-terminal region of the human LAMP protein, which includesa signal sequence domain and an intra-organelle stabilizing domain. Thenucleic acid further provides sequences for an encoded chimeric proteinthat comprises the peanut allergen polyprotein AraH1/AraH2/AraH3. Thenucleic acid further includes sequences encoding a portion of theC-terminal region of the human LAMP protein, which includes atransmembrane region and a targeting region. The nucleotide sequence forthe coding region of the chimeric protein is provided as SEQ ID NO: 8The chimeric protein encoded by the vector of FIG. 6A is presentedschematically in FIG. 6B (and as SEQ ID NO:9).

The domains present in the nucleic acids of the invention are describedin more detail below with respect to the functions provided by theencoded chimeric proteins. It is to be understood that practice of theinvention is not dependent upon or limited by any particular nucleicacid or protein sequence, but rather it is the combination of elementsand domains that provides the advantages and properties to theconstructs. It is also to be understood that the description relating tothe various domains of the nucleic acid construct, when discussed in thecontext of the physical and functional characteristics of the encodedprotein, and vice versa. It is sufficient to apprise one of skill in theart of the physical and functional characteristics of either the nucleicacids or the proteins. It is a simple matter using computers and thedegeneracy of the genetic code to arrive at all possible nucleic acidmolecules encoding known protein sequences and to arrive at proteinsencoded by nucleic acids. Thus reference to a physical or functionalcharacteristic of a particular protein sequence immediately discloses tothe skilled artisan all of the possible nucleic acid sequencesassociated with that physical or functional characteristic, and viceversa.

It is also well within the skill of those of skill in the art to designand combine two or more nucleic acid molecules or sequences to arrive ata sequence encoding a chimeric protein according to the invention.Likewise, it is well within the skilled artisan's abilities to selectand combine transcription and translation control elements to expressthe coding sequences and chimeric proteins in vivo or in vitro asdesired. Accordingly, these commonly used techniques need not bediscussed in detail herein to enable one to practice the presentinvention.

The nucleic acid of the invention comprises a signal sequence domain.The signal sequence domain contains a signal sequence that is providedfor insertion of the encoded chimeric protein into a biological membranethat defines the border between an external environment and an internalenvironment. The signal sequence also directs transfer of the proteinfrom the external environment to the internal environment. The generalstructure of a signal sequence is well known in the art, as are numerousexamples of particular signal sequences. The practitioner is free toselect any appropriate signal sequence according to the variousselection parameters for each embodiment falling within the scope ofthis invention. In exemplary embodiments, the signal sequence is onethat directs the chimeric protein to the endoplasmic reticulum. It isimportant to note at this juncture that the signal sequence domain isthe only portion of the chimeric protein that contains a signalsequence. As such, the naturally-occurring signal sequences of allergensresiding in the allergen domain have been removed prior to inclusion ofthe allergen sequences in the construct. It has been found that removalof these individual signal sequences improves the overall performance ofthe construct in vivo.

The nucleic acid of the invention comprises an intra-organellestabilizing domain (IOSD). The IOSD comprises a sequence that encodes anamino acid sequence that binds, via chemical bonds, to one or moresequences in the allergen domain and protects those sequences fromdegradation (e.g., proteolysis) prior to arrival of the chimeric proteinin the endosomal/lysosomal compartment. In essence, the IOSD can beenvisioned as a protective cap for the allergen domain sequences,shielding those sequences, and in particular allergenic epitopesequences, from proteolytic enzymes, low pH, and otherprotein-destabilizing substances and conditions. The IOSD can be any ofa number of known or engineered sequences, including, but not limitedto, a LAMP polypeptide lumenal domain and the macrosialin/CD68 protein,which is a heavily glycosylated transmembrane protein that is expressedin macrophages and macrophage-like cells as a late endosomal protein.The key feature of the IOSD is the ability of the IOSD to bind to andprotect the allergen domain from proteolysis until the MHC class IImolecule is released from the invariant peptide. In this way, thethree-dimensional structures of the allergenic epitope(s) are preserveduntil active MHC class II molecules are available for interaction. Inpreferred embodiments, the IOSD comprises all or part of the sequence ofa lysosomal protein. In some embodiments, the IOSD is a protein orpolypeptide other than a LAMP polypeptide lumenal domain, such as, butnot limited to, macrosialin/CD68.

The nucleic acid construct of the invention comprises an allergendomain. The allergen domain comprises one or more sequences that encodeallergen proteins, polypeptides, or peptides, which comprise one or moreallergenic epitopes. The allergen domain does not include signalsequences from the allergens present. Numerous proteinaceous allergensare known in the art, and any one or combination of allergens and/orallergenic epitopes can be used in accordance with the presentinvention. Where less than a full-length allergenic sequence is used,preferably, one or more epitopes of the full-length allergen protein areprovided in the context of their natural positions within the allergenicprotein. More specifically, the present invention provides for improvednucleic acid vaccines, in which the vaccines encode chimeric proteinsthat retain or substantially retain their three dimensional structureuntil MHC class II molecules are competent to bind to epitopes on thechimeric proteins. In this way, an improved immune response can beelicited, as compared to delivery to the MHC class II molecules of shortpeptides, which generally will lack appropriate three dimensionalstructures. Accordingly, it is preferred that the allergen domain encoderelatively long amino acid sequences that include one or more epitopes,if originally present on the allergen protein.

The allergen domain can include two or more allergens, each containingone or more allergenic epitopes. It is known that certain allergenicproteins contain two or more epitopes. As a preferred embodiment of theinvention uses an entire allergenic coding region (i.e., the codingregion lacking a signal sequence), or a substantial portion thereof, ofan allergenic protein, certain allergen domains will include two or moreepitopes in their naturally-occurring relationship. Alternatively, twoor more known epitopes can be fused into one coding region. Yet again,in exemplary embodiments, two or more allergenic proteins, or allergenicregions thereof, are present in the allergen domain. Where two or moreepitopes are engineered to be present in a single epitope domain, theepitopes can be from the same antigenic protein. Alternatively, they canbe from two different proteins of the same species. Yet again, they canbe from the same protein of two different species. Furthermore, they canbe from two or more different proteins from two or more differentspecies. In essence, any combination of epitopes from the same ordifferent proteins from the same or different species is contemplated bythis invention. Likewise, the order of the various allergens andepitopes can be varied in any way imaginable. The mixing of allergenicproteins and/or allergenic peptides from multiple species allows thecreation of a robust nucleic acid vaccine that can provide treatment forallergies to a single source organism (e.g., particular species of tree)based on multiple allergens, as well as treatment for allergies tomultiple source organisms (e.g., multiple plants that release sporesduring the same season of the year) based on multiple allergens. Theability to combat multiple allergies from a single nucleic acid vaccinehas not been proven to date.

The nucleic acid construct of the invention further comprises atransmembrane domain. Transmembrane domains are well known and wellcharacterized physical and functional elements of proteins that existpartially on both sides of a biological membrane. In essence, atransmembrane domain is a linear sequence of amino acids that aregenerally hydrophobic or lipophilic in nature and which function toanchor a protein at a biological membrane. Generally, such sequences are20-25 residues in length. Those of skill in the art are well aware ofsuch sequences and can easily obtain or engineer a suitabletransmembrane sequence for use in the present invention.

In addition to the elements discussed above, the nucleic acid of theinvention comprises a targeting domain. The targeting domain is asequence that encodes an amino acid sequence that functions to targetthe encoded chimeric protein to the endosomal/lysosomal compartment.While not so limited in its identity, in preferred embodiments, thetargeting domain comprises the C-terminal cytoplasmic targeting sequenceof the LAMP polypeptide, DC-LAMP, LAMP2, LAMP-3, LIMP II, ENDOLYN, ormacrosialin/CD68.

In embodiments, the nucleic acid of the invention comprises, as part ofthe allergen domain, the sequence of SEQ ID NO:2 (i.e., the Cry J2nucleotide sequence lacking its signal sequence) or another sequenceencoding SEQ ID NO: 3 (i.e., the Cry J2 protein sequence lacking itssignal sequence) in the allergen domain. SEQ ID NO:2 consists ofnucleotides encoding the full protein coding sequence of Cry J2, withthe exception of its signal sequence (i.e., SEQ ID NO:2), a pectatelysase protein found in the pollen of Cryptomeria japonica. Cry J2 iswell known in the art to be correlated with seasonal and persistentallergies in areas where cedar pollen is present. IgE specific to Cry J2is commonly found in allergic patients in areas near cedar groves. It isto be noted that, in the Sequence Listing provided as part of thedisclosure of the invention, the signal sequence for each allergen, ifpresent, is noted. It is to be understood that, within the context ofthe constructs of the invention, these signal sequences are not present.

In other embodiments, the nucleic acid comprises the sequence of SEQ IDNO:4 (i.e., the Cry J1 nucleotide sequence, lacking its signal sequence)or another sequence encoding SEQ ID NO:5 (i.e., the Cry J2 proteinsequence lacking its signal sequence). In yet other embodiments, thenucleic acid comprises the sequence of both SEQ ID NO:2 and SEQ ID NO:4,or other sequences encoding SEQ ID NO:3 and SEQ ID NO:5, respectively.In embodiments, the nucleic acid comprises one or more of the othersequences disclosed herein, such as those encoding any of the followingallergens: Cry J3 (Cry J3.8; C. japonica; SEQ ID NO: 10; signal sequenceis residues 1-26), CJP-4 (C. japonica; SEQ ID NO: 11), CJP-6 (C.japonica; SEQ ID NO: 12), CJP-8 (C. japonica; SEQ ID NO: 13; signalsequence is residues 1-35), CPA63 (C. japonica; SEQ ID NO: 14; signalsequence is residues 1-20), CJP38 (C. japonica; SEQ ID NO: 15; signalsequence is residues 1-28), Cha o 1 (C. obtuse; SEQ ID NO: 16; signalsequence is residues 1-21), Jun a 1 (J. ashei; SEQ ID NO: 17; signalsequence is residues 1-21), Jun v 1 (J. virginiana: SEQ ID NO: 18;signal sequence is residues 1-21), Cup a 1 (H. arizonica; SEQ ID NO: 19;signal sequence is residues 1-21), Jun o 1 (J. oxycedrus; SEQ ID NO:20;signal sequence is residues 1-21), Cup s 1 (C. sempervirens; SEQ IDNO:21; signal sequence is residues 1-21) Cha o 2 (C. obtuse; SEQ IDNO:22; signal sequence is residues 1-22), Jun a 2 (J. ashei; SEQ IDNO:23; signal sequence is residues 1-22), Cup a 2 (H. arizonica; SEQ IDNO:24), Jun a 3 (J. ashei; SEQ ID NO:25; signal sequence is residues1-16), Jun r 3 (J. rigida; SEQ ID NO:26; signal sequence is residues1-26), Cup s 3 (C. sempervirens; SEQ ID NO:27; signal sequence isresidues 1-26), Cup a 3 (H. arizonica; SEQ ID NO:28), Ch4A (P.monticola; SEQ ID NO:29; signal sequences is from residues 1-25), Ch4-1(P. menziesii; SEQ ID NO:30; signal sequence is residues 1-26), PT-1 (P.taeda; SEQ ID NO:31), and LTP (P. abies; SEQ ID NO:32; signal sequenceis from residues 1-25). Nucleic acid and amino acid sequences not listedwith reference to SEQ ID NOs are also publicly available. It is a merematter of computer program implementation to arrive at protein sequencesaccording to the present invention based on the nucleic acid sequences.Of course, biochemically homologous sequences to these protein sequencesare encompassed by these embodiments. For example, sequences showing 30%or more identity, such as 40% or more, 50% or more, 75% or more, 90% ormore 95% or more, 98% o or more, or 99% or more to the disclosedsequences are encompassed by these embodiments. It is to be understoodthat this concept applies not only to the particular sequences ofallergens disclosed herein, but to all protein and nucleic acidsequences provided herein. Further, as stated above, each value withinthe disclosed ranges are understood to be specifically encompassed bythe present disclosure.

In a particular instance of the invention, a DNA vaccine comprising SEQID NO:2 or another sequence encoding SEQ ID NO: 3 within the allergendomain is provided. When such a vaccine is administered to a patient forwhom there is considerable evidence of a Japanese red cedar allergy, thevaccine results in the de novo synthesis of a fusion or chimeric (theseterms used interchangeably herein) protein comprising the allergen CryJ2 (presented within SEQ ID NO:3). Due to the combination of domainspresent on the chimeric protein, the protein is directed from theendoplasmic reticulum into the endolysosomal pathway, resulting in theprocessing of the fusion protein into epitopes in MHC vesicles, some ofwhich become bound to MHC class II molecules, leading to an enhancedhumoral immune response.

In another instance of the invention, a DNA vaccine comprising thesequence of SEQ ID NO:4 or another sequence encoding SEQ ID NO:5 withinthe allergen domain is provided. When such a vaccine is administered toa patient for whom there is considerable evidence of a Japanese redcedar allergy, the vaccine results in the de novo synthesis of a fusionor chimeric (these terms used interchangeably herein) protein comprisingthe allergen Cry J1 (found within the sequence of SEQ ID NO:4). Due tothe combination of domains present on the chimeric protein, the proteinis directed from the endoplasmic reticulum into the endolysosomalpathway, resulting in the processing of the fusion protein into epitopesin MHC vesicles, some of which become bound to MHC class II molecules,leading to an enhanced humoral immune response.

In another instance of the invention, a DNA vaccine comprising SEQ IDNO:6 within the allergen domain is provided. When such a vaccine isadministered to a patient for whom there is considerable evidence of aJapanese red cedar allergy, the vaccine results in the de novo synthesisof a fusion or chimeric (these terms used interchangeably herein)protein comprising the allergens CryJ1 and Cry J2 (SEQ ID NO:7). Due tothe combination of domains present on the chimeric protein, the proteinis directed from the endoplasmic reticulum into the endolysosomalpathway, resulting in the processing of the fusion protein into epitopesin WIC vesicles, some of which become bound to WIC class II molecules,leading to an enhanced humoral immune response.

In another instance of the invention, a nucleic acid encoding the fullprotein coding sequence of Jun a1, a pectate lysase belonging to thegenus Juniperus ashei, is provided in the allergen domain. Jun a1demonstrates a high degree of sequence identity with Cry J1 and bothretains a similar enzymatic activity to Cry J1 and possesses a highsimilarity in known epitopes.

Other polypeptides are well known to be cross-reactive to Cry J1 andthat this cross-reactivity is due to shared epitopes related to theenzymatic activity of pectate lysase family polypeptides. The familyincludes the major allergen of Japanese cypress (Chamaecyparis obtusa(Ch o1)), and includes allergens from: Juniperus ashei (Jun a 1),Juniperus virginiana (Jun v 1), Cuppressus arizonica (Cup a 1),Juniperus oxycedrus (Jun o 1), and Cupressus sempervirens (Cup s 1). Ithas been observed in the literature that there is strongcross-reactivity among allergic patients to pollen from the cedar family(Cupressus). Table I, below, depicts a table showing levels ofcross-reactivity among related proteins. While the invention isdescribed in detail with regard to Cry J1 and Cry J2, it is to beunderstood that one or more of the allergens disclosed herein andparticularly in Table I can be used in addition to or as alternatives tothe Cry J1 and Cry J2 sequences.

TABLE 1 Cryptomeria japonica cross-reactivity to other allergensCryptomeria Cha Jun Jun Cup Jun Cup Cha Jun Cup Jun Jun Cup Cup Japonicao1 a1 v1 a1 o1 s1 o2 a2 a2 a3 r3 S3 A3 Cry J1 80% 79% 80% 75% 85% 85%Cry J2 74% 71% 80% Cry J3 86% 87% 85% 87% Cryptomeria Japonica Ch4ACh4-1 PT-1 LTP CJP-4 70% 57% CJP-6 74% CJP-8 45% CPA63

It is well known in the art that certain sites for inserting anucleotide sequence for a coding region into the nucleotide sequence fora different coding region (i.e., fusion sites) are more favorable thanothers. The location of the allergen sequence taught in, for example,FIGS. 1-5 is taught as the favorable location for using the compositiontaught in the present invention. It is within the scope of the presentinvention to move the location of the allergen sequence to otherlocations, such as within the luminal domain of a LAMP polypeptide orother intra-organelle stabilizing domain. However, it is preferred thatthe allergen is not placed within the coding region of either thetransmembrane or cytoplasmic domain. In a preferred instance of theinvention, the allergen sequence is inserted into the luminal domain ofa LAMP polypeptide within 5 amino acids from the junction with thetransmembrane domain and up to 20 amino acids on the 5′ N terminal sideof a LAMP polypeptide luminal domain.

The nucleic acid of the invention can be provided as a purified orisolated molecule. The nucleic acid also can be provided as part of acomposition. The composition can consist essentially of the nucleicacid, meaning that the nucleic acid is the only nucleic acid in thecomposition suitable for expression of a coding sequence. Alternatively,the composition can comprise a nucleic acid of the invention. Inexemplary embodiments, the composition is a pharmaceutical compositioncomprising the nucleic acid of the invention along with one or morepharmaceutically acceptable substances or carriers. In some embodiments,the composition comprises a substance that promotes uptake of thenucleic acid by a cell. In some embodiments, the composition comprises atargeting molecule that assists in delivering the nucleic acid to aspecific cell type, such as an immune cell (e.g., and APC). Inembodiments, the nucleic acid is part of a delivery vehicle or deliveryvector for delivery of the nucleic acid to a cell or tissue.

In a particular instance of the invention, the composition comprises amixture of two DNA vaccines, where one vaccine comprises the sequence ofone allergen and where the other vaccine comprises the sequence ofanother allergen. The two vaccine constructs can be mixed together at aratio of 1:1, 1:2, 1:3, 1:4, sequentially up to 1:10. The preferredratio is 1:1.

In a particular instance of the invention, the nucleic acids of Cry J1,Cry J2, and/or Jun a2 are present within a nucleic acid delivery vector.In a preferred embodiment of the invention, the nucleic acid deliveryvector does not contain an antibiotic resistance gene, such as thenucleic acid delivery vector taught by U.S. patent applicationpublication number 2008/006554, or vectors that are disclosed in orresult from U.S. patent application publication number 2006/003148. In aparticular instance of the invention, the nucleic acid is a viralvector, such as an adenoviral vector.

The nucleic acids and compositions are novel and useful as agents forreducing allergic reactions in patients. For example, the nucleic acidsand compositions are useful in reducing pollinosis in patients with ademonstrated allergic reaction correlated with Japanese red cedarpollen, or from a homologous pollen or allergen. As another non-limitingexample, the nucleic acids are useful for reducing food allergies, suchas allergy to peanuts or other nuts. Delivery of nucleic acids andcompositions to treat pollinosis from Japanese red cedar pollen, suchthat the nucleic acids and compositions transfect an antigen presentingcell, results in an increase in serum levels of immunoglobulin G (IgG)specific to epitopes contained within Cry J1 and/or Cry J2. Thisresponse is useful for the reduction of allergy symptoms. Delivery ofallergens for other allergies, including ragweed, other tree pollens,and foods also result in increase in serum levels of IgG.

Methods of treating subjects in need are also provided by thisinvention. The methods are methods of prophylactically treating ortherapeutically treating a subject suffering from or at risk ofdeveloping an allergic reaction to one or more allergens. The methodcomprises administering to the subject a DNA vaccine according to theinvention in an amount sufficient to cause uptake of and expression ofthe DNA vaccine by an APC. Expression of the DNA vaccine results inpresentation of the encoded allergenic epitope(s) on the APC, anddevelopment of an IgG immune response.

In a particular instance of the invention, SEQ ID NO:2, SEQ ID NO:4,and/or another allergen encoding sequence are administered to a cell. Inpreferred embodiments, the cell is an antigen presenting cell, such as adendritic cell. Preferably, the dendritic cell is a human dendriticcell. The present invention can be administered by methods known in theart to be effective delivery methods for nucleic acid vaccines,including intramuscular injection, subcutaneous injection,electroporation, gene gun vaccination, or liposome-mediated transfer.

This invention provides a formulation useful for the treatment ofpollinosis correlated with Japanese red cedar pollen. It has previouslybeen determined that delivering a DNA plasmid encoding the proteincoding sequence of an allergen to an animal can increase IFN-gammaproduction and lower IL-4 production, which is useful in treatinganimals allergic to the specific allergen. The present inventionprovides an improved DNA vaccine composition for treating patients withan allergy correlated to Japanese red cedar pollen. The fusion proteinof the invention has a specific intracellular trafficking pattern thatintersects with MHC class II vesicles, and results in enhancedpresentation of allergen epitopes to the immune system, specificallyresulting in an enhanced antibody response. Nucleic acids andcompositions provided by the present invention are useful for conductingallergy immunotherapy.

The present invention provides a formulation that when administered to acell results in an increased specific antibody response. The increasedantibody response to the allergen is useful for treating an IgE-mediatedallergic disease. IgE has certain properties related to its cellularrestriction and the resulting intracellular signaling upon bindingcognate allergen. IgE is generated against an allergen when B cellsreceive IL-4 secreted by Th2 cells. This helps instruct B cells toproduce IgE class antibodies. Upon secretion by B cells, IgE binds toFc-eRI, its high affinity receptor expressed by mast cells andeosinophils, resulting in these cells and the animal becoming sensitizedto future allergen exposure. Consequently, the symptoms of allergy canbe triggered upon the ingestion, inhalation, or mucosal contact with anallergen. Due to the binding properties of antibodies, it has beenproposed that one way of reducing allergy symptoms is to chelate freeallergen available for binding by IgE through competition with otherantibody classes. In particular, an allergy formulation that increasesIgG has been proposed to be an pathway for reducing allergic disease.The invention described herein induces enhanced IgG production, thuscausing a decrease in the ratio of IgE to IgG in a clinicallysignificant manner. The results of studies that have been conductedindicate that at day 98, the level of IgG induced by a Cry J2-LAMPconstruct is greater than that induced by delivery of nucleotidesencoding unmodified Cry J2.

In another instance of the invention, a method is taught for selectingpectate lysase polypeptides found in the pollen of a cedar tree, fordetermining the degree of sequence homology with the amino acid ornucleic acid sequence of a Cry J1, a pectate lysase, so that a newcomposition of matter similar to Cry J1 can be generated, and so thatadministration of the homologous composition of matter to a patientwould produce a therapeutic result useful for treating allergiescorrelated with cedar pollen.

EXAMPLES

The invention will now be described with reference to exemplaryembodiments of the invention. The following examples are intended togive the reader a better understanding of the construction and activityof the constructs of the invention, and should not be construed as alimitation on the scope of the invention.

Example 1 General Materials and Methods

Immunizations and Sera Collection

Six to eight week old female BALB/c mice were purchased from HarlanLaboratories, Frederick, Md. and maintained at our animal facility inRockville, Md. The DNA immunizations were given either intramuscularlyor intradermal with 50 ug of plasmid DNA in a volume of 100 ul ofsterile PBS. Sera were obtained by orbital bleed and stored at −20° C.for later analysis. For sensitization, mice were injected with either 5ug/ml of recombinant CRYJ2 (rCRYJ2) or recombinant CRYJ1 (rCYRJ1)together with 100 ul of alum (2 mg/ml) in a total volume of 200 ul. Micewere bled weekly and sera were analyzed for CRYJ specific antibodies byELISA.

Guinea Pigs

Female Guinea pigs were purchased and housed at Spring ValleyLaboratories (Woodline, Md.). The DNA immunizations were givenintramuscularly with 100 ug of plasmid DNA in a volume of 200 ul ofsterile saline. Sera were obtained by cardiac bleed and store at 20° C.for later analysis.

Detection of CYRJ2-Specific Immunoglobulin Responses

Nunc Maxisorp immunoassay plates were coated with rCRYJ2 at aconcentration of 5 ug/ml in PBS overnight at 4° C. After blocking with1% BSA in PBS, sera were diluted in PBS containing 0.05% Tween-20(PBS-T) added and incubated for 1 hour. The IgG, IgG1, or IgG2a bound tothe CRYJ2 immobilized on the wells was detected using peroxidaseconjugated goat anti-mouse IgG, IgG1 or IgG2a antibodies (JacksonLaboratories). TMB substrate (KPL) was added and the enzymatic activitystopped with TMB Stop Solution. The plates were read at 450 nm. In someinstances, Sure Stop Solution (KPL) was used and plates were read at 650nm.

Preparation of Splenocytes for Cytokine Measurements

Spleens were removed aseptically and teased to prepare a single-cellsuspension. To study the primary response, splenocytes were cultured in24-well plates (4×10⁵ cells/well) in the presence or absence of 10ug/ml, 5 ug/ml, or 2.5 ug/ml of rCRYJ2 for 72 hours.

Cytokine Assays

Supernatants were assayed for the presence of IFN-gamma and IL-4 byELISA. Matched antibody pairs were used for IFN-gamma and IL-4 and doneaccording to manufacturer's instructions. The standard curves weregenerated with mouse recombinant IFN-gamma and IL-4. All antibodies andcytokines were purchased from Invitrogen, Carlsbad, Calif. The detectionlimits of IFN-gamma and IL-4 assays were 20 and 10 pg/ml in respective.

Example 2 Expression of Allergens from Constructs

To show that the nucleic acid constructs of the invention can be used toexpress one or multiple allergens in transformed cells, human 293 cellswere transfected with the CryJ2-LAMP plasmid, CryJ1+J2-LAMP plasmid(FIG. 4), CryJ1-LAMP plasmid, CryJ1 plasmid (lacking the CryJ1 signalsequence; FIG. 7), and the base plasmid vector alone (negative control;SEQ ID NO: 1). The results of the experiments are shown in FIG. 9.

FIG. 9A shows the results of the transfection reactions, with detectionusing an anti-Cry J2 antibody. Briefly, thirty micrograms of cell lysatewas electrophoresed, then transferred to a membrane for immunoblotting.Proteins were detected by immunoblotting with a CryJ2 monoclonalantibody, followed by chemiluminescence. As can be seen from the Figure,constructs comprising the CryJ2 allergen alone, and the CryJ1+CryJ2allergens were detected (lanes 2 and 3), whereas other allergens werenot. In this experiment, the naturally-occurring signal sequences forthe CryJ1 and CryJ2 allergens were removed prior to the experiment,except for the construct in lane 5. These results show not only that theconstructs of the invention are suitable for expression of allergens,but also that multiple allergens can be co-expressed.

FIG. 9B shows the results of the transfection reactions, with detectionusing an anti-CryJ1 antibody. Briefly, thirty micrograms of cell lysatewas electrophoresed, then transferred to a membrane for immunoblotting.Proteins were detected by immunoblotting with a CryJ1 monoclonalantibody, followed by chemiluminescence. As can be seen from the Figure,constructs comprising the CryJ1+CryJ2 allergens (lacking natural signalsequences) were detected (lane 3), as was the construct comprising theCryJ1 allergen in which the naturally-occurring signal sequence had beenremoved (lane 5). However, the construct in which the Cry1 allergen,which included its natural signal sequence, was not detected. Theseresults show that the constructs of the invention are suitable forexpression and detection of multiple allergens, and that removal ofnaturally-occurring signal sequences is important in expressing anddetecting products.

Example 3 Data Supporting MHC II Processing Pathway for Constructs

To determine if chimeric proteins produced from the constructs of theinvention are processed through the MHC II pathway, a set of experimentswas performed to compare the immune response to the CryJ2 protein whenadministered as a coding region on a plasmid or as an allergen domain ona construct according to the present invention. The results arepresented in FIGS. 10A and 10B.

More specifically, the figure shows the CryJ2 specific responsefollowing four DNA immunizations and crude pollen extract sensitization.Groups of mice (n=5) were immunized subcutaneously with eitherCRYJ2-LAMP plasmid DNA or CRYJ2 plasmid (see FIG. 8) DNA on days 0, 7,14, and 21. Six weeks (day 77) following the last DNA immunization, themice were sensitized with crude pollen extract in alum and given abooster three weeks (day 91) later. The data show the values generatedfrom the pooled sera for each time point. IgG1 (FIG. 10A) and IgG2a(FIG. 10B) response in mice receiving CRYJ2-LAMP DNA remained elevatedthrough day 112 and well above those mice that received CRYJ2 plasmidDNA that did not include LAMP. Delivery of allergens by way ofconstructs according to the invention thus provide a superior MHC IIresponse than delivery of allergens without the context of theconstructs of the invention.

Example 4 Dosing Rationales—Comparison of Immune Response to DifferentDoses of Constructs and to Vector Alone

FIG. 11 shows a CryJ2-specific response following four DNA immunizationsat different levels of dosing, for both IgG2a production and IgG1production. Groups of mice (n=5) were given either 10 ug, 50 ug, or 100ug of CRYJ2-LAMP plasmid DNA or Vector DNA intramuscularly on days 0, 7,14, and 21. Three weeks following the last DNA immunization, the micewere sacrificed and spleens removed for Cytokine Induction assays.

The data show the values generated from the pooled sera for each vaccinedose. All three concentrations of CRYJ2-LAMP plasmid DNA gave IgG1 andIgG2a responses, with the 50 ug dose appearing to have given the highestantibody response. Vector alone, at any of the concentrations, did notinduce any antibody response. These data show that there is adose-dependent response for invoking an immune response, and that theimmune response is, at least in part, an MHC II type response.

Example 5 Further Data Showing an Immune Response Via the MHC II Pathway

In this set of experiments, secretion of cytokines in supernatants ofstimulated spleen cells was determined using IL-4 and IFN-gamma asmarkers. Specifically, spleen cells of mice (n=3) were harvested at day42 and cultured in the presence of 10 ug/ml, 5 ug/ml, 2.5 ug/ml, or norCRYJ2. Spleen cells from naive mice were used as a negative control.IL-4 and IFN-gamma levels of rCRYJ2 stimulated splenocytes were measuredby ELISA in pg/ml.

The data are presented in FIGS. 12A and 12B. The data show that micereceiving 50 ug of CRYJ2-LAMP plasmid DNA had a significantly higherexpression of IFN-gamma (an established biomarker for activation of theMHC II immune response pathway) than those receiving the lower dose ofplasmid DNA. There was very little response seen of IL-4 levels in anyof the groups, an established biomarker for the MHC I pathway. There wasalso very little response, if any, with IL-5 (data not shown). Theseresults indicate that Cry J2-LAMP DNA immunization induced therecruitment of Th1 memory cells and not Th2 cells, as indicated by theproduction of IFN-gamma and not IL-4 after stimulation with therecombinant Cry J2 protein.

Example 6 Studies on the Therapeutic Effect of Immunization withCryJ2-LAMP DNA Vaccine in Previously CryJ2 Sensitized Mice

To study the therapeutic effects of the DNA-LAMP-CryJ2 vaccine, groupsof mice (n=5) were sensitized with three injections of 5 ug of rCRYJ2recombinant protein and four weeks later, treated with four injectionsof CRYJ2-LAMP plasmid DNA given in a weekly (7 day) intervals. The DNAimmunizations induced a booster effect for IgG2a and a transientincrease for IgG1 antibodies indicating a Th1-directed modulatory effectof the DNA vaccine. Two additional DNA immunizations on days 167 and 174boosted the CRYJ2 specific IgG2a response and almost no change in IgG1response. Visual examination of the mice revealed no physical distressor skin reactions. There were also no changes in appetite nor did theyappear lethargic. The effects on IgG1 and IgG2a titers are shown inFIGS. 13A and 13B, respectively.

Example 7 Induction of IFN-Gamma and IL-4 in Mouse Spleen Cell Cultures

The therapeutic effect of CryJ2-LAMP DNA vaccine was also studied interms of cytokine induction. Spleen cells of mice (n=3) were harvestedat day 183 and stimulated with varying concentrations of rCRYJ2. Spleencells from naive mice were used as a negative control. IL-4 andIFN-gamma levels of rCRYJ2 stimulated splenocytes were measured by ELISAin pg/ml. Significantly elevated expression of IFN-gamma was detected inthe CRYJ2-LAMP vaccinated group compared with that in the vector group.However, IL-4 expression showed no difference from the vector group. Theincrease in IFN-gamma as a result of Cry J2-LAMP DNA immunizationpresumably involves the recruitment of antigen-specific Th1 cells andthe creation of a Th1 cytokine milieu. Data obtained from thisexperiment is presented in FIGS. 14A and 14B.

Example 8 Detection of Circulating CryJ2 Protein in Sera

Mice were immunized with Cry J2 protein, pDNA-Cry J2 (no LAMP) and CryJ2-LAMP-vax. Serum samples were taken at days 0, 1, 2, 3, 4, and 7 andevaluated for the presence of free Cry J2 protein in a sensitivesandwich immunoassay. Free Cry J2 was detected in the protein andnon-LAMP immunization. However, no free allergen was detected in anytime point in any experiment with Cry J2-LAMP-vax immunized mice(minimum detectable level 2 ng/ml). Data supporting these statements areprovided in FIG. 12.

LAMP vaccines according to the invention will be the only formulationsthat treat allergies without introducing free allergen into the patientsystemically. This is unlike traditional immunotherapy which cansometimes result in anaphylactic reactions due to systemic introductionof allergen. This experiment shows that mice which received the CryJ2-LAMP DNA plasmid did not have free Cry J2 protein and thus notreleased into the systemic circulation as seen with mice given proteinalone or Cry J2 DNA without LAMP.

Example 9 Effectiveness of DNA Vaccines in Guinea Pigs

To expand the scientific understanding of the function of the presentnucleic acid constructs in other mammals, studies were performed infemale guinea pigs immunized with the CryJ2-LAMP DNA vaccine, thenchallenged with recombinant CryJ2 protein. The results of the studiesare shown in Figure FIGS. 16A and 16B.

Specifically, female guinea pigs received intramuscular injections of100 ug of CRYJ2-LAMP DNA Vaccine or vector alone on days 0, 7, and 14.Four weeks following the last DNA vaccine immunization on day 14, theguinea pigs received subcutaneous injections of 10 ug/ml of rCRYJ2protein/alum on days 42 and 49. Serum samples were obtained from guineapigs on days 0, 21, 35, 63, and 77. The data show that the meanabsorbance values for the guinea pigs receiving CRYJ2-LAMP DNA increasedthrough day 35 for IgG2 with little or no IgG1 response. The increase inIgG2a is consistent with what is typically seen in a Th1 biasedresponse.

Example 10 Further Investigation in Other Mammals—Toxicology DataShowing Safety

New Zealand White rabbits received intramuscular injections of 4.128 mgof CRYJ2-LAMP DNA. Age and gender-matched control rabbits receivedsaline alone. Rabbits were immunized on days 1, 14, 28, 42, and 56.Serum samples were obtained from rabbits on days 1, 14, 28, 42, 56, 58,and 85. Mean absorbance values of rabbit serum at 1:100 followingmultiple IM injections of CryJ2-LAMP plasmid or saline are shown in FIG.17. As can be seen from the Figure, the data show that the meanabsorbance values for the rabbits receiving saline are less than 0.100.The absorbance values of rabbits in the groups treated with CRYJ2-LAMPDNA generally increased through day 42 and in some cases increasedthrough day 85.

Example 11 Applicability to Food Allergies

Over the last 25 years, 8 significant peanut allergens have beenidentified based on sensitization in peanut allergic patients. Threemajor peanut allergens are most commonly recognized by IgE of peanutallergic individuals: 65-100% recognize Ara h1, a 63.5 kDa seed storagevicilin family protein; 71-100% recognize Ara h2, a 17 kDa seed storageconglutin family protein; and 45-95% recognize Ara h3, a 14 kDa seedstorage glycinin family protein. In addition to being a common causativeagent in triggering peanut-dependent allergic reactions and anaphylaxis,these three proteins also appear to promote stronger allergic reactions.Targeting these allergens as the basis for peanut allergy immunotherapyhas the potential of providing the broadest protection from strongallergic reactions among the diverse population of peanut allergies.Phase I clinical trials are currently underway that use hypo allergenicforms of the three major allergens and a heat killed bacterium adjuvantas allergy immunotherapy. This trial is ongoing, but the eventualcommercialization of such a therapy will be a challenge due to a highlycomplex manufacturing process.

To address the rising incidence of food allergies, and in particularpeanut allergy, a nucleic acid construct according to the invention wascreated. The construct is depicted in FIG. 6A, and a schematic of theencoded chimeric protein is depicted in FIG. 6B, as discussed above.This construct can be used to generate a predominantly WIC II responsein subjects to which it is administered. The presence of the three mostcommon peanut allergens in a single chimeric protein allows for a broadimmunization, which will treat the vast majority of peanut allergies inthe population.

The construct was expressed and the results shown in FIG. 18. FIG. 19shows that all three allergens can be expressed and detected as a singlepoly-protein on Western blots.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the practice of the presentinvention and in construction of the nucleic acid constructs withoutdeparting from the scope or spirit of the invention. Other embodimentsof the invention will be apparent to those skilled in the art fromconsideration of the specification and practice of the invention. It isintended that the specification and examples be considered as exemplaryonly, with a true scope and spirit of the invention being indicated bythe following claims.

FURTHER SEQUENCES FOR SEQUENCE LISTING

In addition to the sequences provided in the formal Sequence Listingprovided as part of this application, the following sequences comprisepart of the present disclosure:

-   -   1. The nucleotide sequence of the coding region for the Cry        1-Cry2-LAMP chimeric construct, as follows:

Cry J1 + J2-LAMP SEQ ID NO: 6 ccgcctaatg agcgggcttt tttttcttag ggtgcaaaag gagagcctgt aagcgggcac   60tcttccgtgg tctggtggat aaattcgcaa gggtatcatg gcggacgacc ggggttcgag  120ccccgtatcc ggccgtccgc cgtgatccat gcggttaccg cccgcgtgtc gaacccaggt  180gtgcgacgtc agacaacggg ggagtgctcc ttttggcttc cttccccttc ttccgcttcc  240tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca  300aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca  360aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg  420ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg  480acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt  540ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt  600tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc  660tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt  720gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt  780agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc  840tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa  900agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt  960tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1020acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1080tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1140agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1200tcagcgatct gtctatttcg ttcatccata gttgcctgac tcctgcaaac cacgttgtgg 1260tagaattggt aaagagagtc gtgtaaaata tcgagttcgc acatcttgtt gtctgattat 1320tgatttttgg cgaaaccatt tgatcatatg acaagatgtg tatctacctt aacttaatga 1380ttttgataaa aatcattagg taccccggct ctagatggca tgacattaac ctataaaaat 1440aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 1500cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 1560gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca 1620tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 1680aggagaaaat accgcatcag attggctatt ggccattgca tacgttgtat ccatatcata 1740atatgtacat ttatattggc tcatgtccaa cattaccgcc atgttgacat tgattattga 1800ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 1860gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 1920tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 1980aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 2040caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 2100acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 2160ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 2220gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 2280gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 2340tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 2400gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct 2460cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 2520cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 2580ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 2640agccggctct ccacgctttg cctgaccctg cttgctcaac tctagttctc tcgttaactt 2700aatgagacag atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg 2760tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa gaagacgaag 2820aagacgaaga agacaaaccg tcgtcgacat ggcgccccgc agcgcccggc gacccctgct 2880gctgctactg ctgttgctgc tgctcggcct catgcattgt gcgtcagcag caatgtttat 2940ggtgaaaaat ggcaacggga ccgcgtgcat aatggccaac ttctctgctg ccttctcagt 3000gaactacgac accaagagtg gccctaagaa catgaccctt gacctgccat cagatgccac 3060agtggtgctc aaccgcagct cctgtggaaa agagaacact tctgacccca gtctcgtgat 3120tgcttttgga agaggacata cactcactct caatttcacg agaaatgcaa cacgttacag 3180cgtccagctc atgagttttg tttataactt gtcagacaca caccttttcc ccaatgcgag 3240ctccaaagaa atcaagactg tggaatctat aactgacatc agggcagata tagataaaaa 3300atacagatgt gttagtggca cccaggtcca catgaacaac gtgaccgtaa cgctccatga 3360tgccaccatc caggcgtacc tttccaacag cagcttcagc cggggagaga cacgctgtga 3420acaagacagg ccttccccaa ccacagcgcc ccctgcgcca cccagcccct cgccctcacc 3480cgtgcccaag agcccctctg tggacaagta caacgtgagc ggcaccaacg ggacctgcct 3540gctggccagc atggggctgc agctgaacct cacctatgag aggaaggaca acacgacggt 3600gacaaggctt ctcaacatca accccaacaa gacctcggcc agcgggagct gcggcgccca 3660cctggtgact ctggagctgc acagcgaggg caccaccgtc ctgctcttcc agttcgggat 3720gaatgcaagt tctagccggt ttttcctaca aggaatccag ttgaatacaa ttcttcctga 3780cgccagagac cctgccttta aagctgccaa cggctccctg cgagcgctgc aggccacagt 3840cggcaattcc tacaagtgca acgcggagga gcacgtccgt gtcacgaagg cgttttcagt 3900caatatattc aaagtgtggg tccaggcttt caaggtggaa ggtggccagt ttggctctgt 3960ggaggagtgt ctgctggacg agaacagcct cgaggacaat cctattgatt cctgctggcg 4020tggagattct aactgggcac agaaccggat gaaactggct gactgtgccg tgggctttgg 4080ctcttccact atgggaggga agggaggcga cctgtacact gttacaaaca gcgacgacga 4140ccctgtcaat ccagcacccg gaaccttgag atatggtgca acgcgagacc gaccactttg 4200gatcatcttt agcggaaaca tgaacatcaa gttgaagatg cctatgtaca tagctgggta 4260caaaaccttc gacggcagag gagcccaagt gtacattggc aacggaggtc cctgcgtgtt 4320catcaagcgt gttagtaatg tgatcattca cggtctgcac ctctatggct gttcaacaag 4380cgtgctgggg aatgtgctga tcaatgagtc attcggtgtt gaacccgtgc acccacagga 4440cggtgatgcg ttgacactga ggacagccac caatatctgg attgaccata acagtttctc 4500taacagctca gatggcctgg tggatgtcac cttgagtagc acaggggtca caatcagcaa 4560caatctgttc ttcaaccatc ataaggtgat gctgctgggc cacgacgatg cgtattccga 4620cgataagagc atgaaagtga cggtggcctt taaccagttt ggtcctaact gtggacagcg 4680gatgcctaga gccaggtacg gactggtgca cgtggccaac aacaactatg atccgtggac 4740tatctatgca attggcggtt cttccaaccc gacgatactg agtgaaggga actcctttac 4800cgctcccaat gagagctaca agaagcaggt caccatccgc ataggctgca aaactagttc 4860atcctgtagc aactgggtgt ggcagtccac tcaagatgtc ttctacaacg gagcttactt 4920cgttagcagt gggaaatacg aaggtggcaa catatacaca aagaaagagg ctttcaatgt 4980gagaatggc aatgccactc cccagctcac caagaatgca ggggtgctca cctgctccct 5040gagcaaacgg tgcggcggtg gtggcctcga ggatcagtca gcgcagatca tgctggatag 5100cgtggtggag aagtacctga ggagtaacag gtcactgcgc aaggttgagc attccagaca 5160cgacgctatc aacatcttca acgtggagaa gtacggtgct gtcggagacg ggaagcacga 5220ctgcaccgaa gccttttcta cagcctggca agctgcctgc aagaatccct cagccatgct 5280cctcgtgcct gggtctaaga agtttgtcgt gaataacctt ttcttcaatg gaccctgcca 5340gccacacttt accttcaaag ttgatgggat catcgcagcc tatcagaacc cagctagctg 5400gaagaacaat cggatctggt tgcagtttgc caaactgaca ggattcaccc tgatggggaa 5460aggcgtgatc gacggacagg gcaaacagtg gtgggcaggg cagtgcaagt gggtcaatgg 5520tagggagatt tgcaatgaca gggaccgtcc taccgctatc aagtttgatt tcagcacagg 5580actgattatt caggggttga agctgatgaa tagtccagag tttcaccttg tgtttggcaa 5640ttgtgaaggt gtgaagatca taggcattag cattacagca cctcgcgatt ctcccaatac 5700ggacggcatt gacatcttcg cctccaagaa ctttcacctg caaaagaata ccattggcac 5760aggcgacgac tgcgtggcca ttggcactgg cagcagcaat atcgttatcg aagatttgat 5820atgtggtcct gggcatggca taagcattgg aagcctgggt agagaaaact caagagctga 5880agtcagctat gttcacgtta acggagcgaa gttcattgat acccagaacg gactgcgaat 5940caaaacttgg caagggggaa gtggcatggc atctcacatc atctacgaga acgtcgagat 6000gatcaattcc gagaacccca tactgattaa ccaattctat tgtacttccg cctctgcctg 6060ccagaatcag agatcagccg tgcagattca ggacgtgaca tacaagaata tccgagggac 6120gagcgctacc gctgccgcaa tacagctcaa atgttccgat agcatgccct gcaaagatat 6180caagcttagt gatatctccc tcaaactgac tagcggaaag atagcgtcct gtctcaatga 6240taacgcaaat ggctacttct cagggcatgt gatccctgca tgcaaaaacc ttagcccgag 6300tgcgaaacgc aaagaatcca aatcccataa gcatccgaag actgtgatgg tcgagaacat 6360gagagcctac gacaaaggga accggacgag gattctgctg ggctctcgac cgccaaactg 6420taccaacaaa tgtcacggtt gttctccatg caaagctaaa ctggtgatag tgcatcgcat 6480catgcctcaa gagtactatc cccagcgttg gatttgtagt tgccatggca agatctatca 6540cccagaattc acgctgatcc ccatcgctgt gggtggtgcc ctggcggggc tggtcctcat 6600cgtcctcatc gcctacctcg tcggcaggaa gaggagtcac gcaggctacc agactatcta 6660gtaaggatct ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc 6720tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt 6780 gtctctcact cggaaggaca taagggcggc cgctagc 6817

-   -   2. The nucleic acid sequence of the coding region for he Ara H1        I H2/h3 polyprotein:

SEQ ID NO: 8ccgcctaatg agcgggcttt tttttcttag ggtgcaaaag gagagcctgt aagcgggcac   60tcttccgtgg tctggtggat aaattcgcaa gggtatcatg gcggacgacc ggggttcgag  120ccccgtatcc ggccgtccgc cgtgatccat gcggttaccg cccgcgtgtc gaacccaggt  180gtgcgacgtc agacaacggg ggagtgctcc ttttggcttc cttccccttc ttccgcttcc  240tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca  300aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca  360aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg  420ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg  480acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt  540ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt  600tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc  660tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt  720gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt  780agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc  840tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa  900agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt  960tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1020acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1080tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1140agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1200tcagcgatct gtctatttcg ttcatccata gttgcctgac tcctgcaaac cacgttgtgg 1260tagaattggt aaagagagtc gtgtaaaata tcgagttcgc acatcttgtt gtctgattat 1320tgatttttgg cgaaaccatt tgatcatatg acaagatgtg tatctacctt aacttaatga 1380ttttgataaa aatcattagg taccccggct ctagatggca tgacattaac ctataaaaat 1440aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 1500cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 1560gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca 1620tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 1680aggagaaaat accgcatcag attggctatt ggccattgca tacgttgtat ccatatcata 1740atatgtacat ttatattggc tcatgtccaa cattaccgcc atgttgacat tgattattga 1800ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 1860gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 1920tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 1980aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 2040caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 2100acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 2160ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 2220gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 2280gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 2340tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 2400gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct 2460cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 2520cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 2580ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 2640agccggctct ccacgctttg cctgaccctg cttgctcaac tctagttctc tcgttaactt 2700aatgagacag atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg 2760tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa gaagacgaag 2820aagacgaaga agacaaaccg tcgtcgacat ggcgccccgc agcgcccggc gacccctgct 2880gctgctactg ctgttgctgc tgctcggcct catgcattgt gcgtcagcag caatgtttat 2940ggtgaaaaat ggcaacggga ccgcgtgcat aatggccaac ttctctgctg ccttctcagt 3000gaactacgac accaagagtg gccctaagaa catgaccctt gacctgccat cagatgccac 3060agtggtgctc aaccgcagct cctgtggaaa agagaacact tctgacccca gtctcgtgat 3120tgcttttgga agaggacata cactcactct caatttcacg agaaatgcaa cacgttacag 3180cgttcagctc atgagttttg tttataactt gtcagacaca caccttttcc ccaatgcgag 3240ctccaaagaa atcaagactg tggaatctat aactgacatc agggcagata tagataaaaa 3300atacagatgt gttagtggca cccaggtcca catgaacaac gtgaccgtaa cgctccatga 3360tgccaccatc caggcgtacc tttccaacag cagcttcagc aggggagaga cacgctgtga 3420acaagacagg ccttccccaa ccacagcgcc ccctgcgcca cccagcccct cgccctcacc 3480cgtgcccaag agcccctctg tggacaagta caacgtgagc ggcaccaacg ggacctgcct 3540gctggccagc atggggctgc agctgaacct cacctatgag aggaaggaca acacgacggt 3600gacaaggctt ctcaacatca accccaacaa gacctcggcc agcgggagct gcggcgccca 3660cctggtgact ctggagctgc acagcgaggg caccaccgtc ctgctcttcc agttcgggat 3720gaatgcaagt tctagccggt ttttcctaca aggaatccag ttgaatacaa ttcttcctga 3780cgccagagac cctgccttta aagctgccaa cggctccctg cgagcgctgc aggccacagt 3840cggcaattcc tacaagtgca acgcggagga gcacgtccgt gtcacgaagg cgttttcagt 3900caatatattc aaagtgtggg tccaggcttt caaggtggaa ggtggccagt ttggctctgt 3960ggaggagtgt ctgctggacg agaacagcct cgagaagtcc agcccctacc agaagaaaac 4020cgagaacccc tgcgcccagc ggtgcctgca gtcttgtcag caggaacccg acgacctgaa 4080gcagaaggcc tgcgagagcc ggtgcaccaa gctggaatac gaccccagat gcgtgtacga 4140ccctagaggc cacaccggca ccaccaacca gagaagccct ccaggcgagc ggaccagagg 4200cagacagcct ggcgactacg acgacgacag acggcagccc agaagagaag agggcggcag 4260atggggacct gccggcccta gagagagaga acgcgaggaa gattggagac agcccagaga 4320ggactggcgg aggccttctc accagcagcc ccggaagatc agacccgagg gcagagaagg 4380cgagcaggaa tggggcacac ctggctctca cgtgcgcgag gaaaccagcc ggaacaaccc 4440cttctacttc ccctcccggc ggttcagcac cagatacggc aaccagaacg gccggatcag 4500agtgctgcag agattcgacc agcggagccg gcagttccag aacctgcaga accaccggat 4560cgtgcagatc gaggccaagc ccaacaccct ggtgctgccc aaacacgccg acgccgacaa 4620catcctcgtg atccagcagg gccaggccac cgtgacagtg gccaacggca acaacagaaa 4680gagcttcaac ctggacgagg gccacgccct gagaatcccc agcggcttca tcagctacat 4740cctgaacaga cacgacaatc agaacctgag ggtggccaag atcagcatgc ccgtgaacac 4800ccctggccag ttcgaggact tcttccccgc atcctcccgg gaccagagca gctacctgca 4860gggcttcagc cggaataccc tggaagccgc cttcaacgcc gagttcaacg agatcagacg 4920ggtgctgctg gaagagaacg ctggcggaga gcaggaagaa cggggccaga gaagatggtc 4980caccagaagc agcgagaaca acgagggcgt gatcgtgaag gtgtccaaag aacacgtgga 5040agaactgacc aagcacgcca agagcgtgtc caagaagggc tccgaggaag agggggacat 5100caccaacccc atcaatctga gagagggcga gcccgacctg agcaacaact tcggcaagct 5160gttcgaagtg aagcccgaca agaagaaccc ccagctgcag gacctggaca tgatgctgac 5220ctgcgtggaa atcaaagagg gggccctgat gctgccacac ttcaactcca aagccatggt 5280catcgtggtc gtgaacaagg gcaccggcaa cctggaactg gtggccgtgc ggaaagagca 5340gcagcagaga ggccgcagag aggaagaaga ggacgaggac gaagaagaag agggatccaa 5400ccgggaagtg cggcggtaca ccgccagact gaaagaaggc gacgtgttca tcatgcctgc 5460cgcccacccc gtggccatca atgcctctag cgagctgcat ctgctgggct tcggcattaa 5520cgccgagaac aatcaccgga tctttctggc cggcgacaaa gacaacgtga tcgaccagat 5580cgagaagcag gccaaggacc tggcctttcc cggctctggc gaacaagtgg aaaagctgat 5640caagaaccag aaagaaagcc acttcgtgtc cgccagaccc cagagccagt ctcagagccc 5700tagctccccc gagaaagagt ctcctgagaa agaggaccag gaagaggaaa accagggcgg 5760caagggccct ctgctgagca tcctgaaggc cttcaatggc ggcggaggca ggcagcagtg 5820ggaactgcag ggcgacagaa gatgccagtc ccagctggaa cgggccaacc tgaggccttg 5880cgagcagcac ctgatgcaga aaatccagcg cgacgaggac agctacggcc gggatcctta 5940cagccccagc caggaccctt actcccctag ccaggatccc gacagaaggg acccctacag 6000ccctagcccc tacgatagaa gaggcgccgg aagcagccag caccaggaaa gatgctgcaa 6060cgagctgaac gagtttgaga acaaccagcg ctgcatgtgc gaggccctgc agcagatcat 6120ggaaaatcag agcgaccggc tgcagggacg gcagcaggaa cagcagttca agagagagct 6180gcggaacctg ccccagcagt gtggactgag agccccccag agatgcgacc tggaagtgga 6240aagcggcggc agagatcggt acggcggagg gggcgtgacc ttcagacagg gcggagaaga 6300gaatgagtgc cagtttcagc ggctgaacgc ccagaggccc gacaacagaa tcgagagcga 6360gggcggctac atcgagacat ggaaccccaa caaccaggaa tttcagtgcg ctggggtggc 6420cctgagcagg accgtgctga gaagaaatgc cctgaggcgg cccttctaca gcaacgcccc 6480cctggaaatc tacgtgcagc agggcagcgg ctacttcggc ctgatctttc ccggatgccc 6540ctccacctat gaggaacccg ctcaggaagg cagacggtat cagagccaga agcctagcag 6600acggttccaa gtgggccagg acgatcccag ccaacagcag caggactctc accagaaggt 6660gcaccgcttc gacgagggcg acctgatcgc tgtgccaacc ggcgtggcct tctggatgta 6720caacgacgag gataccgacg tcgtgaccgt gaccctgagc gacaccagct ccatccacaa 6780ccagctggac cagttcccca ggcggtttta cctggccggc aatcaggaac aggaatttct 6840gagataccag cagcagcagg gctccagacc ccactacaga cagatcagcc ctagagtgcg 6900gggcgacgaa caggaaaatg agggcagcaa catcttctcc ggctttgccc aggaatttct 6960gcagcacgcc ttccaggtgg accggcagac cgtggaaaac ctgagaggcg agaacgagag 7020agaggaacag ggcgccatcg tgactgtgaa gggcggcctg aggatcctga gccccgacga 7080agaggatgag tcctctagaa gcccccccaa ccgccgggaa gagttcgatg aggaccgcag 7140cagacctcag cagcggggga agtacgacga gaacaggcgg ggctacaaga acggcatcga 7200ggaaacaatc tgcagcgcca gcgtgaagaa gaatctgggc cggtccagca accccgacat 7260ctacaatcca caggccggca gcctgcggag cgtgaacgaa ctggatctgc ccatcctggg 7320atggctgggc ctgtctgccc agcacggcac catctaccgg aacgccatgt tcgtgcctca 7380ctacaccctg aatgcccaca ccatcgtggt ggctctgaac ggccgcgccc acgtccaagt 7440ggtggacagc aacggcaatc gggtgtacga tgaagaactg caggaaggac acgtcctggt 7500ggtgccccag aattttgccg tggccgccaa ggcccagtcc gagaactatg agtatctggc 7560cttcaagacc gacagccggc cctctatcgc caatcaagcc ggcgagaaca gcatcatcga 7620caacctgccc gaggaagtgg tggccaacag ctaccggctg cctagagagc aggcccggca 7680gctgaagaac aacaaccctt tcaagttctt cgtgccccca ttcgaccacc agagcatgag 7740agaggtggcc gaattcacgc tgatccccat cgctgtgggt ggtgccctgg cggggctggt 7800cctcatcgtc ctcatcgcct acctcgtcgg caggaagagg agtcacgcag gctaccagac 7860tatctagtaa ggatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg 7920agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 7980ttttgtgtct ctcactcgga aggacataag ggcggccgct age 8023The amino acid sequence of the coding region for the Ara H1 I H2 I H3polyprotein chimeric construct, as follows:

AraH-LAMP SEQ ID NO: 9 <220> <221> SIGNAL <222> (1)..(27) <220> <221>N-LAMP <222> (28)..(380) <220> <221> AraH1 <222> (383)..(983) <220><221> AraH2 <222> (988)..(1138) <220> <221> AraH3 <222> (1143)..(1634)<220> <221> TM/CYTO <222> (1637)..(1672) <400> 7Met Ala Pro Arg Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu Leu1         5            10         15Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Ala Met Phe Met Val          20      25          30Lys Asn Gly Asn Gly Thr Ala Cys Ile Met Ala Asn Phe Ser Ala Ala     35          40          45Phe Ser Val Asn Tyr Asp Thr Lys Ser Gly Pro Lys Asn Met Thr Leu  50            55            60Asp Leu Pro Ser Asp Ala Thr Val Val Leu Asn Arg Ser Ser Cys Gly65          70           75           80Lys Glu Asn Thr Ser Asp Pro Ser Leu Val Ile Ala Phe Gly Arg Gly         85          90           95His Thr Leu Thr Leu Asn Phe Thr Arg Asn Ala Thr Arg Tyr Ser Val      100            105          110Gln Leu Met Ser Phe Val Tyr Asn Leu Ser Asp Thr His Leu Phe Pro     115          120         125Asn Ala Ser Ser Lys Glu Ile Lys Thr Val Glu Ser Ile Thr Asp Ile  130            135          140Arg Ala Asp Ile Asp Lys Lys Tyr Arg Cys Val Ser Gly Thr Gln Val145           150           155         160His Met Asn Asn Val Thr Val Thr Leu His Asp Ala Thr Ile Gln Ala        165           170           175Tyr Leu Ser Asn Ser Ser Phe Ser Arg Gly Glu Thr Arg Cys Glu Gln      180           185           190Asp Arg Pro Ser Pro Thr Thr Ala Pro Pro Ala Pro Pro Ser Pro Ser    195            200          205Pro Ser Pro Val Pro Lys Ser Pro Ser Val Asp Lys Tyr Asn Val Ser  210           215            220Gly Thr Asn Gly Thr Cys Leu Leu Ala Ser Met Gly Leu Gln Leu Asn225         230            235          240Leu Thr Tyr Glu Arg Lys Asp Asn Thr Thr Val Thr Arg Leu Leu Asn          245          250         255Ile Asn Pro Asn Lys Thr Ser Ala Ser Gly Ser Cys Gly Ala His Leu        260          265           270Val Thr Leu Glu Leu His Ser Glu Gly Thr Thr Val Leu Leu Phe Gln    275          280            285Phe Gly Met Asn Ala Ser Ser Ser Arg Phe Phe Leu Gln Gly Ile Gln   290         295          300Leu Asn Thr Ile Leu Pro Asp Ala Arg Asp Pro Ala Phe Lys Ala Ala305           310           315          320Asn Gly Ser Leu Arg Ala Leu Gln Ala Thr Val Gly Asn Ser Tyr Lys         325          330            335Cys Asn Ala Glu Glu His Val Arg Val Thr Lys Ala Phe Ser Val Asn      340           345          350Ile Phe Lys Val Trp Val Gln Ala Phe Lys Val Glu Gly Gly Gln Phe     355          360           365Gly Ser Val Glu Glu Cys Leu Leu Asp Glu Asn Ser Leu Glu Lys Ser  370           375          380Ser Pro Tyr Gln Lys Lys Thr Glu Asn Pro Cys Ala Gln Arg Cys Leu385          390          395       400Gln Ser Cys Gln Gln Glu Pro Asp Asp Leu Lys Gln Lys Ala Cys Glu         405          410          415Ser Arg Cys Thr Lys Leu Glu Tyr Asp Pro Arg Cys Val Tyr Asp Pro       420           425         430Arg Gly His Thr Gly Thr Thr Asn Gln Arg Ser Pro Pro Gly Glu Arg    435           440          445Thr Arg Gly Arg Gln Pro Gly Asp Tyr Asp Asp Asp Arg Arg Gln Pro   450         455           460Arg Arg Glu Glu Gly Gly Arg Trp Gly Pro Ala Gly Pro Arg Glu Arg465          470          475           480Glu Arg Glu Glu Asp Trp Arg Gln Pro Arg Glu Asp Trp Arg Arg Pro          485            490           495Ser His Gln Gln Pro Arg Lys Ile Arg Pro Glu Gly Arg Glu Gly Glu       500           505            510Gln Glu Trp Gly Thr Pro Gly Ser His Val Arg Glu Glu Thr Ser Arg    515          520            525Asn Asn Pro Phe Tyr Phe Pro Ser Arg Arg Phe Ser Thr Arg Tyr Gly  530           535          540Asn Gln Asn Gly Arg Ile Arg Val Leu Gln Arg Phe Asp Gln Arg Ser545         550           555            560Arg Gln Phe Gln Asn Leu Gln Asn His Arg Ile Val Gln Ile Glu Ala          565         570          575Lys Pro Asn Thr Leu Val Leu Pro Lys His Ala Asp Ala Asp Asn Ile        580         585          590Leu Val Ile Gln Gln Gly Gln Ala Thr Val Thr Val Ala Asn Gly Asn     595         600            605Asn Arg Lys Ser Phe Asn Leu Asp Glu Gly His Ala Leu Arg Ile Pro  610          615          620Ser Gly Phe Ile Ser Tyr Ile Leu Asn Arg His Asp Asn Gln Asn Leu625           630            635          640Arg Val Ala Lys Ile Ser Met Pro Val Asn Thr Pro Gly Gln Phe Glu         645            650           655Asp Phe Phe Pro Ala Ser Ser Arg Asp Gln Ser Ser Tyr Leu Gln Gly      660            665          670Phe Ser Arg Asn Thr Leu Glu Ala Ala Phe Asn Ala Glu Phe Asn Glu     675          680           685Ile Arg Arg Val Leu Leu Glu Glu Asn Ala Gly Gly Glu Gln Glu Glu    690          695           700Arg Gly Gln Arg Arg Trp Ser Thr Arg Ser Ser Glu Asn Asn Glu Gly705          710           715          720Val Ile Val Lys Val Ser Lys Glu His Val Glu Glu Leu Thr Lys His          725           730          735Ala Lys Ser Val Ser Lys Lys Gly Ser Glu Glu Glu Gly Asp Ile Thr        740          745           750Asn Pro Ile Asn Leu Arg Glu Gly Glu Pro Asp Leu Ser Asn Asn Phe     755           760          765Gly Lys Leu Phe Glu Val Lys Pro Asp Lys Lys Asn Pro Gln Leu Gln   770          775          780Asp Leu Asp Met Met Leu Thr Cys Val Glu Ile Lys Glu Gly Ala Leu785          790          795         800Met Leu Pro His Phe Asn Ser Lys Ala Met Val Ile Val Val Val Asn        805           810           815Lys Gly Thr Gly Asn Leu Glu Leu Val Ala Val Arg Lys Glu Gln Gln       820            825        830Gln Arg Gly Arg Arg Glu Glu Glu Glu Asp Glu Asp Glu Glu Glu Glu    835          840           845Gly Ser Asn Arg Glu Val Arg Arg Tyr Thr Ala Arg Leu Lys Glu Gly  850          855           860Asp Val Phe Ile Met Pro Ala Ala His Pro Val Ala Ile Asn Ala Ser865         870            875            880Ser Glu Leu His Leu Leu Gly Phe Gly Ile Asn Ala Glu Asn Asn His          885         890            895Arg Ile Phe Leu Ala Gly Asp Lys Asp Asn Val Ile Asp Gln Ile Glu        900          905          910Lys Gln Ala Lys Asp Leu Ala Phe Pro Gly Ser Gly Glu Gln Val Glu    915           920          925Lys Leu Ile Lys Asn Gln Lys Glu Ser His Phe Val Ser Ala Arg Pro  930           935          940Gln Ser Gln Ser Gln Ser Pro Ser Ser Pro Glu Lys Glu Ser Pro Glu945            950           955           960Lys Glu Asp Gln Glu Glu Glu Asn Gln Gly Gly Lys Gly Pro Leu Leu         965          970         975Ser Ile Leu Lys Ala Phe Asn Gly Gly Gly Gly Arg Gln Gln Trp Glu        980          985           990Leu Gln Gly Asp Arg Arg Cys Gln Ser Gln Leu Glu Arg Ala Asn Leu     995          1000                 1005Arg Pro Cys Glu Gln His Leu Met Gln Lys Ile Gln Arg Asp Glu   1010       1015           1020Asp Ser Tyr Gly Arg Asp Pro Tyr Ser Pro Ser Gln Asp Pro Tyr  1025         1030       1035Ser Pro Ser Gln Asp Pro Asp Arg Arg Asp Pro Tyr Ser Pro Ser  1040          1045          1050Pro Tyr Asp Arg Arg Gly Ala Gly Ser Ser Gln His Gln Glu Arg  1055          1060          1065Cys Cys Asn Glu Leu Asn Glu Phe Glu Asn Asn Gln Arg Cys Met  1070          1075          1080Cys Glu Ala Leu Gln Gln Ile Met Glu Asn Gln Ser Asp Arg Leu  1085          1090          1095Gln Gly Arg Gln Gln Glu Gln Gln Phe Lys Arg Glu Leu Arg Asn  1100          1105          1110Leu Pro Gln Gln Cys Gly Leu Arg Ala Pro Gln Arg Cys Asp Leu  1115          1120          1125Glu Val Glu Ser Gly Gly Arg Asp Arg Tyr Gly Gly Gly Gly Val  1130         1135           1140Thr Phe Arg Gln Gly Gly Glu Glu Asn Glu Cys Gln Phe Gln Arg  1145         1150           1155Leu Asn Ala Gln Arg Pro Asp Asn Arg Ile Glu Ser Glu Gly Gly  1160         1165         1170Tyr Ile Glu Thr Trp Asn Pro Asn Asn Gln Glu Phe Gln Cys Ala  1175           1180        1185Gly Val Ala Leu Ser Arg Thr Val Leu Arg Arg Asn Ala Leu Arg   1190         1195          1200Arg Pro Phe Tyr Ser Asn Ala Pro Leu Glu Ile Tyr Val Gln Gln   1205         1210          1215Gly Ser Gly Tyr Phe Gly Leu Ile Phe Pro Gly Cys Pro Ser Thr   1220         1225          1230Tyr Glu Glu Pro Ala Gln Glu Gly Arg Arg Tyr Gln Ser Gln Lys   1235         1240          1245Pro Ser Arg Arg Phe Gln Val Gly Gln Asp Asp Pro Ser Gln Gln   1250         1255          1260Gln Gln Asp Ser His Gln Lys Val His Arg Phe Asp Glu Gly Asp   1265         1270         1275Leu Ile Ala Val Pro Thr Gly Val Ala Phe Trp Met Tyr Asn Asp   1280         1285         1290Glu Asp Thr Asp Val Val Thr Val Thr Leu Ser Asp Thr Ser Ser   1295        1300           1305Ile His Asn Gln Leu Asp Gln Phe Pro Arg Arg Phe Tyr Leu Ala   1310          1315          1320Gly Asn Gln Glu Gln Glu Phe Leu Arg Tyr Gln Gln Gln Gln Gly   1325        1330         1335Ser Arg Pro His Tyr Arg Gln Ile Ser Pro Arg Val Arg Gly Asp   1340         1345            1350Glu Gln Glu Asn Glu Gly Ser Asn Ile Phe Ser Gly Phe Ala Gln   1355         1360         1365Glu Phe Leu Gln His Ala Phe Gln Val Asp Arg Gln Thr Val Glu  1370          1375         1380Asn Leu Arg Gly Glu Asn Glu Arg Glu Glu Gln Gly Ala Ile Val   1385        1390         1395Thr Val Lys Gly Gly Leu Arg Ile Leu Ser Pro Asp Glu Glu Asp   1400         1405            1410Glu Ser Ser Arg Ser Pro Pro Asn Arg Arg Glu Glu Phe Asp Glu   1415        1420             1425Asp Arg Ser Arg Pro Gln Gln Arg Gly Lys Tyr Asp Glu Asn Arg   1430          1435         1440Arg Gly Tyr Lys Asn Gly Ile Glu Glu Thr Ile Cys Ser Ala Ser   1445        1450            1455Val Lys Lys Asn Leu Gly Arg Ser Ser Asn Pro Asp Ile Tyr Asn   1460          1465         1470Pro Gln Ala Gly Ser Leu Arg Ser Val Asn Glu Leu Asp Leu Pro   1475        1480            1485Ile Leu Gly Trp Leu Gly Leu Ser Ala Gln His Gly Thr Ile Tyr   1490         1495         1500Arg Asn Ala Met Phe Val Pro His Tyr Thr Leu Asn Ala His Thr   1505         1510         1515Ile Val Val Ala Leu Asn Gly Arg Ala His Val Gln Val Val Asp   1520          1525         1530Ser Asn Gly Asn Arg Val Tyr Asp Glu Glu Leu Gln Glu Gly His   1535        1540          1545Val Leu Val Val Pro Gln Asn Phe Ala Val Ala Ala Lys Ala Gln   1550         1555         1560Ser Glu Asn Tyr Glu Tyr Leu Ala Phe Lys Thr Asp Ser Arg Pro   1565         1570         1575Ser Ile Ala Asn Gln Ala Gly Glu Asn Ser Ile Ile Asp Asn Leu   1580         1585         1590Pro Glu Glu Val Val Ala Asn Ser Tyr Arg Leu Pro Arg Glu Gln   1595         1600         1605Ala Arg Gln Leu Lys Asn Asn Asn Pro Phe Lys Phe Phe Val Pro   1610         1615         1620Pro Phe Asp His Gln Ser Met Arg Glu Val Ala Glu Phe Thr Leu   1625         1630         1635Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly Leu Val Leu Ile   1640         1645         1650Val Leu Ile Ala Tyr Leu Val Gly Arg Lys Arg Ser His Ala Gly   1655         1660          1665 Tyr Gln Thr Ile   1670

What is claimed:
 1. An isolated or purified nucleic acid moleculeencoding a chimeric AraH-LAMP protein comprising, in sequential order: anucleic acid sequence encoding a signal sequence of a human LAMPprotein; a nucleic acid sequence encoding a lumenal domain of a humanLAMP protein; a nucleic acid sequence encoding AraH1, AraH2 and AraH3,wherein the AraH1, AraH2 and AraH3 nucleic acid sequence does notinclude a naturally-occurring signal sequence of AraH1, AraH2 or AraH3;a nucleic acid sequence encoding a transmembrane domain of a human LAMPprotein; and a nucleic acid sequence encoding a targeting domain of ahuman LAMP protein.
 2. The nucleic acid molecule of claim 1, wherein thechimeric AraH-LAMP protein consists of an amino acid sequence which isat least 90% identical to the amino acid sequence of SEQ ID NO:
 9. 3.The nucleic acid molecule of claim 1, wherein the chimeric AraH-LAMPprotein consists of an amino acid sequence of SEQ ID NO: 9 in which 10or less amino acids are substituted, deleted, inserted and/or added. 4.The nucleic acid molecule of claim 1, wherein the chimeric AraH-LAMPprotein consists of an amino acid sequence of SEQ ID NO:
 9. 5. Thenucleic acid molecule of claim 1, wherein the nucleic acid moleculecomprises DNA.
 6. An expression vector comprising the nucleic acidmolecule of claim
 5. 7. A pharmaceutical composition comprising theexpression vector of claim
 6. 8. The pharmaceutical composition of claim7 further comprising a pharmaceutically acceptable carrier.
 9. A methodof treating or preventing a peanut allergy in a subject in need thereof,the method comprising administering to the subject the nucleic acidmolecule of claim 1 in an amount effective to elicit an immune response.